text:
Real-time kinetic measurements of the interactions between lactogenic hormones and prolactin-receptor extracellular domains from several species support the model of hormone-induced transient receptor dimerization.

Interactions of recombinant soluble prolactin receptors-extracellular domains (PRLR-ECDs) from rabbit, rat, and cow and human growth hormone receptor ECD with immobilized human growth hormone, several prolactins, and bovine placental lactogen were studied utilizing surface plasmon resonance. This method enables real-time kinetic measurements of the interactions and calculations of kinetic constants and of the stoichiometry of interaction, even in cases where only transient interactions occur. In contrast to gel filtration or crystallographic studies, where in most cases the interaction of PRLR-ECDs with various lactogenic hormones indicated formation of 1:1 complexes, our surface plasmon resonance experiments indicated in all cases the transient formation of a 2:1 complex. In most of the interactions the 2:1 complex was very unstable and underwent rapid dissociation to a 1:1 complex. This situation was particularly characteristic of homologous interactions involving hormone and receptor from the same species and was mainly attributed to increased dissociation constants. We suggest that as in the case of growth hormone PRLR activation occurs via hormone-induced transient homodimerization of the receptor, lasting only a few seconds, and that this may be sufficient to initiate the biological signal. Once the signal is initiated, the receptor dimer is no longer required. Its rapid dissociation to a 1:1 complex or to its components may even be advantageous in that it permits activation of additional receptors.

Prolactin (PRL) 1 and growth hormone (GH) receptors (R) belong to the large cytokine receptor family, which is characterized by a conserved 200 -240-amino acid sequence in the extracellular domain (ECD) (1)(2)(3). Four positionally conserved cysteine residues that form two disulfide bridges and a WSXWS motif are found, respectively, in the amino-and carboxyl-terminal portions of the ECD. Accumulating evidence indicates that ligand-induced homodimerization, heterodimerization, or oligomerization of the receptor is a common step that leads to the activation of the cytoplasmic, receptor-associated Jak tyrosine kinases (4,5). However, despite the structural homology of the ligands, characterized by a bundle of four ␣-helices (6), and the ECDs of their receptors, the mechanism and stoichiometry of receptor oligomerization does not seem to follow identical or even similar patterns (7)(8)(9).
The most extensively studied interaction to date has been that between human (h) GH and hGHR-ECD. Using a slightly truncated, soluble, nonglycosylated hGHR-ECD (amino acids 1-238), de Vos and co-workers (10) crystallized the hormonereceptor complex, proving the simultaneous binding of the hormone to two hGHR-ECDs and structurally identified the different hGH sites involved in the interactions. Results of these studies substantiated those of Wells and co-workers (11)(12)(13), who were the first to suggest a hormone-induced sequential receptor homodimerization in which hGH binds one molecule of hGHR-ECD through site I, and then a second through site II, and offered a paradigm of ligand-induced receptor oligomerization in the cytokine receptor family (14). These results were further confirmed by us using full-size (amino acids 1-246) hGHR-ECD (15). Similar bovine (b) GH-induced homodimerization using recombinant soluble bGHR-ECD was also reported (16). Indirect evidence reported by us (17,18) suggested that receptor dimerization or oligomerization also occurs in PRLR as an initial step in hormone-induced signal transduction. Thus, to evaluate whether the same paradigm of hormone-receptor interaction applies to PRL, we prepared soluble recombinant PRLR-ECDs from rabbit (rb) (19,20), bovine (b) (21), and rat (r) (22) and studied their interactions with several PRLs and related lactogenic hormones in binding and gel filtration experiments. Results of those studies revealed a highly heterogeneous picture. Whereas with bPRLR-ECD, only 1:1 ECD-hormone complex could be detected (21), with rbPRLR-ECD, most of the hormones reacted similarly with the exception of bovine placental lactogen (bPL) (20) and ovine (o)PL (23), in which a 2:1 stoichiometry was found. In contrast to the interactions of rPRLR-ECD with hGH, oPRL, bPL (22), and oPL, 2 which yielded a 2:1 stoichiometry, homologous rPRL formed only a 1:1 complex. A similar 2:1 stoichiometry for the complex between rPRLR-ECD expressed in mammalian cells and oPRL was also reported (24). Moreover, we found that homodimerization of rPRLR-ECD by hGH, and even more so by oPRL, is concentration-dependent, and upon dilution from micromolar to nM concentrations, the complex dissociated from a 2:1 to a 1:1 form (22). Recent crystallographic studies of the complex between hGH and hPRLR-ECD also revealed a 1:1 stoichiometry and documented the formation of the complex through site I of hGH (25). Furthermore, our research indicated that despite the differences in the binding properties and stoichiometries of interactions of the respective PRLR-ECDs and lactogenic hormones, the biopotencies of these hormones, as determined in several homologous or heterologous in vitro bioassays, were, surprisingly, almost identical (26 -28).
The present work attempts to clarify this situation by making use of a novel method for real-time kinetic measurements of the interactions between macromolecules, based on surface plasmon resonance (29). This technique enables a determination of kinetic constants and the stoichiometry of interaction of a complex in which one component is immobilized on a flexible dextran matrix, whereas the other is free in solution. It has been used successfully for studies of hGH (30), epidermal growth factor (31), and interleukin 2 (32) receptors. Using this method, we detected weak dimeric complexes that had not been identified by classical binding, gel filtration, or crystallographic studies, and the kinetics of their formation and dissociation was assessed. Preliminary results of this study were reported at the Endocrine Society meeting (33).
Coupling of hGHR-ECD and rbPRLR-ECD to Dextran Matrix via Thiol Groups -Covalent linking of hGHR-ECD and rbPRLR-ECD was performed according to Johnsson et al. (29). The immobilization process was performed with 25 mM Hepes buffer, pH 7.4, containing 150 mM NaCl and 0.05% (w/v) P20 surfactant (HBS) at a flow rate of 5 l/min. The matrix was activated for 3 min with 0.05 M EDC/NHS in HBS, followed by an 8-min injection of 80 mM 2-(2-pyridinyldithio)ethanamine hydrochloride in 0.1 M sodium borate buffer, pH 8.5, and then rbPRLR-ECD (50 g/ml) in 5 mM sodium borate buffer, pH 5, for 5 min, or hGHR-ECD (70 g/ml) in 5 mM sodium borate buffer, pH 4.7, for 7 min. Unreacted matrix groups were deactivated through a 6-min injection of a 50 mM L-cysteine, 1 M NaCl solution in 0.1 M sodium formate buffer, pH 4.3. This procedure ensured an immobilization of 4000 RU for hGHR-ECD and 800 RU for rbPRLR-ECD. Binding capacities of the immobilized R-ECDs were checked by sequential injections of 2.5 M hormone solutions in HBS for 6 min followed by a regeneration pulse of 4.5 M MgCl 2 for 1 to 2 min.
Coupling of Various Hormones to a CM-Dextran Matrix via Amino Groups-The hormones were covalently linked according to Johnsson et al. (29). HBS was injected at 5 l/min, and activation with 0.05 M EDC/NHS in HBS was carried out for 7-8 min. The various hormones were then injected at concentrations ranging from 50 to 100 g/ml in 10 mM sodium acetate buffers at pH values of 4.5-5, based on the optimal electric charge for electrostatic preconcentration on the matrix surface, preservation of the R-ECD binding capacities for the appropriate levels of hormone immobilization: hGH, pH 4.5 (1400 RU), oPRL, pH 4.7 (3100 RU), rbPRL, pH 4.7 (6800 RU), rPRL, pH 4.7 (2500 RU), bPL, pH 5 (5400 RU), and hPRL, pH 5 (3900 RU). Unreacted sites were blocked with an 8-min injection of 1 M ethanolamine hydrochloride at pH 8.5. Binding capacities were checked by repeated injections of 5 M R-ECDs in HBS. All immobilized hormones except rbPRL could be regenerated over 50 runs with 4.5 M MgCl 2 pulses (1-2 min). Rabbit PRL was regenerated by washing out the bound R-ECDs with HBS for 20 min at 20 l/min.
Presentation of hGH by Anti-hGH mAb Capture-Rabbit anti-mouse IgG-purified immunoglobulins were covalently linked to the matrix gel via amino-group coupling as recommended by Pharmacia Biosensor. Two anti-hGH mAbs (33) were used as capture antibodies: mAb 10A7, which binds hGH at a distance from both receptor interaction sites, and mAb 7B11 which binds to site II of hGH, thereby allowing the receptor to bind only to site I. Each of the mAbs prepared in HBS at 100 g/ml was captured by a 6-min injection at a flow rate of 5 l/min, which ensured immobilization of 2000 RU on rabbit anti-mouse IgG-purified immunoglobulins. For R-ECD-hGH interaction studies, saturating levels (2.5 M) of hGH were injected for 6 min, and about 500 RU of the hormone were thus captured in each case. For kinetic characterization of hGH capture by the two mAbs, serial runs were performed with 0 -1000 nM hormone solution. Regeneration at the end of each run was achieved by a 2-min pulse of 100 mM HCl.
Kinetic Measurements of R-ECD-Hormone Interactions-All experiments were performed at a flow rate of 5 l/min in HBS at 25°C. Once the hormone being tested was immobilized, either covalently through amino-group coupling or via mAb capture (for hGH), serial dilutions ranging from 1000 to 62.5 nM of each R-ECD were injected for 6 min and then washed out for 10 min prior to regeneration. Because the recombinant R-ECDs were lyophilized with sodium bicarbonate buffers at a salt:protein ratio of 1:2 ratio, bulk refractive indexes varied with sample dilution and were corrected by injections of the same dilutions into flow cells where unrelated ligands had been immobilized. In experiments in the presence of Zn 2ϩ , hormones were immobilized in HBS as described earlier. Then the 15 M ZnCl 2 was added to the HBS running buffer, and all subsequent dilutions were performed in 15 M ZnCl 2 -containing HBS.
Data Analysis and Calculation of Kinetic Constants-BIAcore incorporated software (BIA Evaluation and BIA Simulation) allowed us to 1) fit experimental curves with 1:1 or 1:2 association/dissociation models and calculate the probabilities of either one of these being the most accurate representation of reality, and 2) calculate kinetic constants with standard deviations; reverse verification of calculated data was performed by simulating the interaction assuming a variable relative occupation of sites.
Theoretical Analysis-The solution concentration of the receptor at time t is denoted R(t), whereas its concentration in the flowing solution is R o . Eventually, i.e. in a steady state, R(t) ϭ R o . The hormone can exist in any of the four states H(0,0), H(1,0), H(0,1) and (H1,1), which denote, respectively, the concentrations of hormone molecules without bound receptors, or with one receptor at site I or site II or sites I and II. The set of nonlinear differential equations describing the process of receptor binding is given in Appendix I. Schematically, in the case of independent binding sites, the binding reactions considered are: The depleted concentration of the receptor is continuously replenished due to the flow, but depending on hormone concentration, the flow rate, and the reaction rate constants, R(t) may be severalfold smaller than R o . We assumed that the interaction follows a sequential pattern, whereby H(0,1) cannot be formed from H(0,0), i.e. the forward direction in Equation 2 is ignored, but H(0, 1) can be formed by dissociation of H(1,1), i.e. the reverse reaction in Equation 4. The steady state solutions turn out to be independent of the flow rate and are the same as those obtained by assuming constant receptor concentrations. For the
The association phase was followed through 600 s after which the infusion of the soluble PRLR-ECDs was stopped, and the dissociation phase in continuous buffer flow was monitored for additional 600 s.
case of independent sites we found an analytic solution for the steady state stage: in which the bars indicate the fractions of hormones in the different states and K I ϭ k I, on /k I, off and K II ϭ k II, on /k II, off . These equations show that in the limit of R o 3 0, H(0,0) ϭ 1, whereas the other quantities vanish, and when R o tends to infinity, H(1,1) ϭ 1 and the other quantities vanish. The steady state solutions are independent of hormone concentration.

RESULTS
Optimization of the Immobilization Procedure-Initially, rb, r, and bPRLR-ECDs were immobilized via their single free cysteine residue. Despite reasonably good covalent binding (2000 -3000 RU/mm 2 ), the immobilized ECDs totally lost their ability to bind to the different PRLs, bPL, or hGH. Alternative immobilization of PRLR-ECDs using a cross-linker acting through amino groups also failed, and the binding capacity of the immobilized ECDs was drastically reduced. This most likely happened because of heterogeneous immobilization via  several amino groups, some of them probably being important for hormone binding. An alternative protocol was employed to overcome this difficulty; the respective hormones were immobilized via their amino groups. This method yielded promising results; in all cases, the immobilized hormones retained a high capacity of interaction with soluble PRLR-ECDs or, in the case of hGH, with hGHR-ECD. As shown in Tables I-IV, the immobilized hormones were capable of binding the soluble R-ECDs with apparent stoichiometries ranging from 1:1 to 1:2, under conditions in which the ligands were applied at 0.25-1 M.

Interaction of rb, b, and rPRLR-ECDs with Several
Lactogenic Hormones-The interactions of rbPRLR-ECD with oPRL and hGH yielded similar kinetic parameters ( Fig. 1A and Table  I). The apparent stoichiometry indicated a 1:1 interaction at equilibrium. However, analysis of the kinetic data by fitting to a theoretical model (Fig. 2) showed to be unsuited to the 1:1 model and strongly favoring the two-site model. Similar results were also obtained with hPRL; although here, the apparent stoichiometry of interaction was 2:1. Interaction with rPRL also yielded a 2:1 apparent stoichiometry, but in this instance the k on values were ϳ10-fold higher than those of aforementioned complexes. The interaction with bPL was much stronger, mainly due to a relatively low k off for site II, and an apparent 2:1 complex was observed by both SPR and gel filtration analysis (20). As can be seen, the interaction of rbPRLR-ECD with the putative site I of all hormones was always much stronger than with site II, although the relative affinities varied to a large extent. The lower affinity for site II unanimously originated from elevated k off values, whereas the k on values had hardly any effect. In contrast, the interaction with the homologous hormone rbPRL was characterized by a rather weak interaction with both sites I and II.
Interaction of bPRLR-ECD with oPRL, bPL, and hGH indicated formation of an apparent 1:1 complex as determined by both SPR (Fig. 1B and Table II) and gel filtration (21). However, analysis of association and dissociation kinetics, performed as shown in Fig. 2 (data not presented), indicated a complete lack of fit to the presumed one-site model and a very good fit to the two-site model. Simulation analysis also favored the 50/50 double-site model with the exception of hGH. As in the binding of hGH, the different affinities for sites I and II could be attributed to the 10 -20-fold higher k off values for site II, whereas the differences in the k on values were relatively small. Similar to the aforementioned case of homologous interaction, the interaction of bPRLR-ECD with oPRL (identical to bPRL) and bPL, both homologous hormones, was also characterized by a relatively lower affinity of the homologous hormones, not only for site II but also for site I. Here again, the lower affinity resulted mainly from the increase in k off .
The interaction of rPRLR-ECD with different hormones (Fig.  1C and Table III) yielded in SPR experiments an apparent stoichiometry of 2:1, except with rPRL in which only a 1:1 stoichiometry has been observed, as also shown in gel filtration experiments (22). Kinetic analysis revealed a very good fit to the two-site model and a poor fit to the one-site model. All kinetic parameters also gave an optimal 50/50 two-site simulation analysis. Bovine PL exhibited the highest affinity to both sites I and II, although the latter was relatively lower, mainly due to a 5-fold higher k off value as compared with site I. Other hormones, such as h or oPRL, exhibited lower affinities for binding to both sites, again mainly due to higher k off values. The affinity of rPRLR-ECD for the homologous rPRL was weaker than for other hormones. Whereas the affinities and kinetic constants for site I resembled those of oPRL and hPRL, the affinity to site II was 6 -15-fold lower, primarily because of a lower k on .
Presentation of hGH by Anti-hGH mAbs-Direct, covalent immobilization of the hormones via their amino groups raised the question of whether the immobilization yielded homogeneous or heterogeneous populations. The latter could affect the calculation of association and dissociation kinetic constants. To answer this question, an alternative method of homogeneous immobilization of hGH was employed using two anti-hGH mAbs, mAb 10A7 which binds to hGH in a way that does not interfere with the latter's ability to form a 1:2 homodimeric complex with hGHR-ECD, and mAb 7B11 which interacts with an epitope located within site II of hGH and, as such, enables only a 1:1 interaction through site I (35). Both mAbs were captured by a covalently immobilized rabbit anti-mouse IgGpurified immunoglobulins. The association of hGH with mAb 7B11 was ϳ20-fold slower than with 10A7 (k on ϭ 0.98 ϫ 10 4 and 2.05 ϫ 10 5 mol Ϫ1 s Ϫ1 , respectively), and the dissociation was also ϳ3-fold slower (k off ϭ 4.5 ϫ 10 Ϫ5 and 14.1 ϫ 10 Ϫ5 s Ϫ1 , respectively). The resultant K d/ values for mAbs 7B11 and 10A7 were, correspondingly, 4.6 and 0.68 nM and their respective calculated half-lives were 257 and 82 min.
Binding of Soluble hGHR-ECD and rPRLR-ECDs to hGH Immobilized via Anti-hGH mAbs-Direct immobilization of hGH versus its presentation via mAb 10A7 yielded very similar results in terms of its interaction with hGHR-ECD ( Fig. 3 and Table IV). The kinetics of association and dissociation were   groups (upper panel), presented via capture of mAb 107A (middle panel) or via capture of mAb 7B11 (bottom panel). In experiments in which hGH was presented via capture of mABs the association phase was preceded by a step of successive capture of the respective mAb and hGH. For other details see the legend to Fig. 1. analyzed using both 2:1 and 1:1 models of interaction. The results did not fit the one-site model with either direct or indirect immobilization, whereas a good statistically significant (p Ͻ 0.05) fitting was obtained with the two-site model. Moreover, calculating the kinetic constants by BIAcore simulation analysis also confirmed the 50/50 double-site model.

FIG. 3. Association and dissociation kinetics between hGHR-ECD and hGH covalently linked to CM-dextran through amino
A comparison of the kinetic and thermodynamic constants for the binding of rb, b, and rPRLR-ECDs by hGH that was either directly immobilized or presented via mAb 10A7 yielded a high degree of similarity, as found with hGHR-ECD (see Fig.  4 Tables I-III). In the case of rbPRLR-ECD ( Fig. 4 and Table I), the interaction with site I of hGH yielded almost identical kinetic constants, whereas the association to site II was enhanced ϳ10-fold in hGH presented via mAb 10A7. Although the apparent stoichiometry was 1:1 in both cases, as in gel filtration experiments (19,20), neither the association nor the dissociation data fit the 1:1 model. In contrast, analysis using the two-site model yielded a much better fit. The discrepancy between the apparent 1:1 stoichiometry and the putative 2:1 stoichiometry (obtained by calculation) can most likely be attributed to the 3 to 4 orders of magnitude difference in the corresponding K d values for the interaction with sites I and II of the hormone. These differences indicate that the interaction with site II is less stable, as determined by measuring the k off constants; correspondingly, the half-life of the complex formed by interaction with site II of hGH was extremely short (Ͻ0.2 min). Similar results were also obtained with bPRLR-ECD ( Fig. 4 and Table II), although in this case the dissociation rate of the complex formed with hGH presented by mAb 10A7 was ϳ5-fold higher than that with the directly immobilized hormone. Here again, an apparent 1:1 stoichiometry was observed, whereas kinetic analysis favored the 2:1 model. Similarly no differences were observed in the interaction of rPRLR-ECD with hGH immobilized directly or indirectly via both mAbs ( Fig. 4 and Table III). In this case, however, not only was the stoichiometry calculated from the kinetic data 2:1, but the apparent stoichiometry was also 2:1, as found previously in gel filtration experiments (22).

and the four top lines in
Presentation of hGH via mAb 7B11 resulted in the formation of 1:1 complex with all three PRLR-ECDs (see Tables I-III, line  5). However, in contrast to the binding of hGHR-ECD in which the kinetic and thermodynamic parameters were close to those calculated for the interaction with site I of the hormone, the corresponding parameters calculated for interaction with b and rPRLR-ECDs were in all cases similar to the values calculated for the interaction with site II. These results indicate that in the latter case, occupation of site II by mAb 7B11 also weakens the apparent interaction of site I of hGH with the respective PRLR-ECDs. This occurs as a result of destabilization of the complex, rather than of changes in the rate of its formation, as evidenced by a ϳ100to 1000-fold increase in k off values. In the case of interaction with rbPRLR-ECD, kinetic constants could not be calculated because the latter's interaction with hGH presented by mAb 7B11 destabilized the mAB⅐hGH complex, causing its dissociation (Fig. 4).
Effect of Zn 2ϩ on the Interaction of hGH with rb, b, and Rat PRLR-ECDs-The cation Zn 2ϩ has been implicated in the interaction of hGH with hPRLR-ECD by forming a coordinate bond between His 19 and Glu 174 of the hormone and Asp 217 and His 218 of the receptor (25). To study whether such an effect also exists in the interaction of hGH with other PRLR-ECDs and hGHR-ECD, binding experiments were carried out in the presence of 15 M ZnCl 2 . The addition of Zn 2ϩ had only a minor effect. With hGHR-ECD, no major change was noted in the dissociation kinetics, but the association was clearly slower (see Table IV, last two lines). With rPRLR-ECD (see Table III, line 6), the addition of Zn 2ϩ increased the dissociation of 2:1 complex to 1:1 complex but rendered the latter more stable. Similar results were obtained with rbPRLR-ECD and bPRLR-ECD (not shown).
Evaluation of "Sequential" Versus "Two Independent Site" Models-Since the BIAcore software does not include the analysis of a sequential binding model, a set of equations aimed at calculating the fractional occupancy of the immobilized hormone by the R-ECD throughout the experiment was developed and utilized to compare the two possible models. A representative experiment in which the fractional occupancy during the first 10 min of interaction was calculated from the respective kinetic constants presented in Table III is shown in Fig. 5. A logarithmic time scale was used for better visualization of the differences occurring during the initial reaction period. The results indicate that the predicted fractional occupancy of sites I and II only differs slightly at the first 2 min, whereby the two independent site model predicts slightly faster simultaneous occupation of both sites than the sequential model. At later times, both models predict almost identical results. Calculation of other interactions yielded a similar picture (not shown).

DISCUSSION
The main question raised in the present work was whether the interaction of various PRLs with their homologous or heterologous receptors leads to the formation of a homodimer, as in a well-documented GH model. Kinetic experiments using three different PRLR-ECDs and several lactogenic hormones strongly suggested that lactogenic hormone-induced PRLR-ECD dimerization does occur as follows: (a) no good fit to the one-site model was found in any case; (b) a good fit enabling a calculation of kinetic rate constants was obtained only using the two-site model; (c) analyzing the kinetic rate constants by BIAcore simulation program confirmed binding to two independent sites, in a 50/50 double-site model. All three criteria were met for all interactions except for that of hGH with bPRLR-ECD, where the fit criteria were indicative of a two-site interaction but the third criterion indicated unequal occupation of sites I and II. The reasons for this exception are unclear. Since the mathematical model presently used in BIAcore software does not allow an analysis of sequential binding, we developed a mathematical model to analyze the results accordingly. We found the difference between the two independent sites model and the sequential model to be rather small, the latter shifting the fractional occupancy curve slightly to the right, especially early on (Fig. 5B). The validity of the sequential model is supported by the findings that (a) hGH immobilized via various mAbs directed to site I is unable to bind any hGHR-ECD, 3   a For Footnotes a-d see Table I. which binds to site II of hGH, did not interfere with binding to site I, as documented in Fig. 3 and Table IV.
The universal feature of a strong binding site (designated site I) and a loose binding site (designated site II), the latter usually exhibiting 5-to 100-fold lower affinity, was observed in most of interactions between lactogenic hormones and PRLR-ECDs studied in this work. Although the present results do not enable topological identification of sites I and II, the recently published three-dimensional structure of hPRLR-ECD⅐hGH complex (25) suggests that the strong binding site I most likely corresponds to the site participating in that interaction, namely the site designated as site I of hGH (10,25). This situation differs from that observed for the hGH-hGHR interaction, in which a stable 2:1 hGHR-ECD⅐hGH complex could be isolated (11,16) and crystallized (10), and most likely indicates formation of transient homodimeric complexes that rapidly dissociate to 1:1 complexes. The only exceptions were interactions of rb and rPRLR-ECDs with bPL, in which the 2:1 complexes were more stable (see Tables I and III). The interaction of rPRLR-ECD with hGH and oPRL also yielded a 2:1 complex, although it was less stable than that formed with bPL (22). These differences parallel the K d II values in Table III. Although no structural interpretation can be made as yet, the extended N-terminal portion of bPL may stabilize the homodimer as truncation of 13 of the N-terminal bPL amino acids has a strongly destabilizing effect (20).
The transient nature of the hormone-receptor complexes was even more pronounced in homologous interactions involving hormones and receptors from the same species. These interactions are generally characterized by faster dissociation of the homodimeric complexes or even the 1:1 complexes (see Tables  I-III), and the differences between the homologous and heterologous interaction can be mainly attributed to higher k off constants. This observation explains the paradoxical situation faced by many investigators of PRLRs who were unable to perform binding experiments using homologous hormones and used hGH or other heterologous lactogenic hormones as the ligand (26 -28). It also explains why the large differences in affinity constants, as calculated by classical binding experiments, were not always paralleled by corresponding differences in biological activity. This seems to be particularly true for the homologous hormones, which often exhibit low thermodynamic association constants (K a ) in contrast to high biological activity (36,37).
The present results strongly suggest that the use of gel filtration or binding experiments as tools for determining binding constants and stoichiometries of interaction may be misleading, since sometimes the 2:1 PRLR-ECD⅐hormone complex cannot be identified due to its rapid dissociation to a 1:1 complex. The use of real-time kinetic analysis based on SPR allows a more accurate interpretation. The currently described progress in the elucidation of PRLR-mediated signal transduction enables us to understand why transient homodimerization of the receptor, lasting only a few seconds, may be sufficient to initiate the biological signal by transphosphorylation of Jak2 or other associated kinases (4,5,38,39). Once these enzymes are activated, the receptor dimer is no longer required. In fact, rapid dissociation of a 2:1 complex to a 1:1 complex or to its components may be advantageous in that it can augment receptor re-availability or permit activation of other receptors. The very recently reported erythropoietin-induced transient homodimerization of erythropoietin receptor, characterized by one high affinity and one low affinity interaction (40), further supports our present suggestion. Our results also have an impact on the strategy for preparing the so-called second generation of recombinant hormones. In the case of hormones that stimulate signal transduction by homo-or heterodimerization of receptors, a high affinity recombinant agonist in which the elevated K a is achieved mainly by lowering the k off will not necessarily be advantageous. Indeed, high affinity analogues of hGH (41) do not exhibit higher biological activity. 4 In contrast, this is not the case when designing hormone antagonists in which prolonged life of the complex is highly beneficial. Parlow (Harbor-UCLA Medical Center, CA) for the gift of ovine, rat, and rabbit prolactins and Dr. Vincent Goffin from Liege, Belgium, for the gift of human prolactin.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
A sulfhydryl oxidase from chicken egg white.

A dimeric glycoprotein containing one FAD per ∼80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 μM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (λmax 560 nm; εobs 2.5 mM−1 cm−1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.

They have been purified from a number of microbial and mammalian sources, but many questions concerning their mechanism and functional roles remain unresolved. Iron-dependent sulfhydryl oxidases have been found in bovine milk (1)(2)(3), in kidney (4), and in pancreatic zymogen granules (5). Coppercontaining sulfhydryl oxidases have been described from kidney and small intestine (6) and skin (7,8).
A second distinct class of sulfhydryl oxidase is provided by FAD-dependent enzymes isolated from both rat seminal vesicles and fungal sources (9 -11). These careful studies show a range of diverse subunit composition and substrate specificity but clearly implicate the flavin cofactor in catalysis (9,10). The present work reports the discovery and characterization of a flavoprotein sulfhydryl oxidase in chicken egg white.
Our study began with the observation that egg white contains not only riboflavin (in the form of riboflavin-binding protein; Ref. 12) but also measurable levels of FAD (13). While the function of riboflavin-binding protein in the transport and storage of vitamin B 2 is well known (14), the reason for the presence of FAD in egg white was obscure. We first purified the FAD-containing protein by following its yellow color and subsequently identified it as a sulfhydryl oxidase active with a wide range of thiol substrates. This paper presents strong evidence for the involvement of a catalytically important disulfide as a second redox-active moiety in these flavin dependent sulfhydryl oxidases. These enzymes therefore show marked functional similarities with the flavin disulfide oxidoreductase family of which glutathione reductase is a prototypical member (15).

EXPERIMENTAL PROCEDURES
Materials-Dithiothreitol, reduced glutathione, cysteine, ␤-mercaptoethanol, and high molecular weight SDS protein standards were from Sigma. Sodium dithionite was from the Virginia Smelting Company.
General Methods-Concentrations of egg white sulfhydryl oxidase are reported with respect to enzyme-bound flavin using a molar extinction coefficient of 12.5 mM Ϫ1 cm Ϫ1 at 454 nm (see below). Visible and ultraviolet absorption spectra were obtained using Perkin-Elmer 552 or HP8452A instruments. Anaerobic procedures, using all-glass titration cuvettes, were as described earlier (16). The oxidase was photoreduced anaerobically 4 cm from a 150-watt flood lamp using 1.2 M 5-deazaflavin and 5 mM EDTA. Maximal EH 2 formation occurred at 90 s, and full reduction required illumination for 6 min. Fluorescence measurements used a Turner model 111 fluorimeter or an Aminco-Bowman Series 2 Luminescence Spectrometer. Unless otherwise stated, all buffers contained 0.3 mM EDTA. Enzyme samples were concentrated and washed using Amicon Centricon 30 ultrafiltration cells.
Assays-Sulfhydryl oxidase was routinely assayed in a Clarke-type oxygen electrode (YSI 5331) and oxygen monitor (YSI 53) using 5 mM DTT 1 in 3 ml of 50 mM potassium phosphate equilibrated with air at pH 7.5, 25°C. A background trace followed the nonenzymatic oxidation of DTT, and assays were started by the addition of enzyme. Thiol substrates were prepared fresh in distilled water and were standardized using DTNB. Concentrated solutions of glutathione were brought to pH 7 prior to standardization. The effect of small levels of disulfide contamination in these samples was evaluated using borohydride treatment as described earlier (9); no significant inhibition of the oxidase by disulfide was noted. Analysis of the stoichiometry of the reaction used 150 M DTT in air-saturated buffer (50 mM phosphate buffer, pH 7.5, 25°C, containing 240 M dissolved oxygen). The addition of 90 nM oxidase led to a 140 M decrease in the concentration of oxygen, and the addition of 20 g of bovine liver catalase returned 72 M oxygen to the solution. The following 100 mM buffers were used for measurement of the pH dependence: sodium acetate (pH 4 -5.5), phosphate (pH 7-8 and 11), Tris/chloride (pH 8 -9), and CHES (pH 10).
Spectrophotometric assays of the purified oxidase used 200 M ferricenium ion and 200 M DTT in 50 mM phosphate buffer, pH 6.0 containing 0.3 mM EDTA. The initial rates were monitored at 300 nm before the precipitation of ferrocene (17). * This work was supported in part by National Institutes of Health Grant GM26643. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Purification of Egg White Sulfhydryl Oxidase-The rdrd strain of single comb White Leghorn chickens (riboflavin-binding protein deficient, see "Results"; Refs. 18 and 19) was maintained on the University of Delaware Farm, and 900 eggs were collected and stored at 4°C. The egg whites were collected (17 liters), and an equal volume of 20 mM Tris buffer, pH 8.1, 4°C was added. The mixture was homogenized by vigorous stirring with a motor-driven impeller, and the pH was adjusted to 8.1 with HCl. The suspension was centrifuged (9 min in a GS3 rotor at 9000 rpm), and the clear supernatants were collected and combined while discarding the gelatinous off-white precipitates. This clarification was necessary for the success of the subsequent batch absorption step. Equilibrated DE52 (Whatman, equivalent to 180 g of damp sorbent) was added to the supernatant, and the suspension was stirred gently for 30 min at 4°C. The yellow ion exchange resin was allowed to settle, collected, and washed with 2 liters of 20 mM Tris, pH 8.1, in a funnel lined with four layers of cheesecloth. The addition of a second aliquot of resin to the supernatant yielded a paler yellow gel, suggesting that all of the flavin was not absorbed by the first batch treatment. However, inclusion of additional batches of gel adversely affects subsequent purification steps, so a single limiting amount of gel was used. The resin was then rinsed with 0.5 liters of 0.5 M NaCl in 20 mM Tris buffer, pH 8.1, 4°C, and the yellow eluate was collected and brought to 45% saturation in ammonium sulfate. The suspension was centrifuged (10,000 rpm, GSA rotor; 10 min), and the clear yellow supernatant was brought to 55% ammonium sulfate. The yellow precipitate was recovered by centrifugation, redissolved in a minimum volume of 20 mM Tris buffer, pH 8.1, 4°C, and dialyzed versus the same buffer. The oxidase in 43% saturated ammonium sulfate was applied to an octyl-Sepharose column (1.5 ϫ 15 cm). The yellow band absorbed to the top of the column was washed at 1 ml/min with 250 ml of 20 mM Tris buffer containing 43% ammonium sulfate. Sulfhydryl oxidase was eluted with a decreasing salt gradient formed by adding 20 mM Tris buffer to a sealed flask containing 100 ml of the 43% ammonium sulfate solution. The enzyme eluted in a narrow band, and fractions with a 280/454 nm absorbance ratio of less than 12-15 were combined, dialyzed versus 20 mM Tris buffer, and concentrated by ultrafiltration. The enzyme was applied in batches to a Superose 12 HR 10/30 FPLC column equilibrated with 75 mM Tris buffer, pH 7.5, 25°C, and fractions with absorbance ratios of less than 10.5 and between 10.5 to 12 were combined separately. Enzyme was concentrated by ultrafiltration and stored frozen in 20 mM Tris buffer, pH 8.1, 4°C, containing 0.3 mM EDTA.
Identification of FAD in Egg White Sulfhydryl Oxidase-Samples of the oxidase in 20 mM Tris buffer, pH 8.1, were denatured either by boiling for 5 min in foil-covered plastic centrifuge tubes or using 88% MeOH. The precipitate was removed by centrifugation, and portions of the supernatant were diluted in water or 10 mM HCl for measurement of flavin fluorescence (20). Solutions of FAD, purified as in Massey and Swoboda (21), and FMN served as controls. The fluorescence of the released flavin increased 4.3-fold in 10 mM HCl, suggesting that sulfhydryl oxidase contained FAD (20). High pressure liquid chromatography of the released flavin on a C18 reverse-phase semipreparative column used a gradient of methanol and 25 mM potassium phosphate, pH 5.3, developed at 2 ml/min: 3 min at 10% methanol; 15-min linear gradient to 60% methanol; 5 min at 60% methanol; 5 min to 10% methanol. In this system, authentic FAD and the flavin released upon denaturation of the oxidase coelute.
Amino Acid Analysis-Samples of untreated and alkylated oxidase were desalted by ultrafiltration against distilled water, and 2 nmol aliquots were hydrolyzed in 6 M HCl for 24 h at 110°C in the presence of 5% phenol. Portions were analyzed with a Beckman 6300 Autoanalyzer equipped with a Sica 7000 integrator (22). For the measurement of cysteine plus half-cystine content, the oxidase (2 M) was denatured in 5.9 M guanidine hydrochloride, incubated with 1 mM DTT for 2 h, and alkylated with 4 mM iodoacetamide for 3 h. Excess reagents were removed by dialysis against two changes of distilled water, and the alkylated protein was lyophilized prior to amino acid analysis. Tryptophan content was estimated spectrophotometrically using the procedure of Edelhoch (23,24). The oxidase was denatured in 6 M guanidine hydrochloride, and the resulting visible spectrum was compared with an equivalent amount of free FAD in denaturant (24). The difference spectrum, representing the contribution of the apoprotein, gave 16 tryptophan residues/subunit.
Carbohydrate Analysis-The oxidase was desalted as above and analyzed for monosaccharide content by Dr. Jeffrey Rohrer, Dionex, Sunnyvale, CA. Samples (25 g of protein) were hydrolyzed using either trifluoroacetic acid for monosaccharides (25) or HCl for sialic acid (26), and the hydrolysates were analyzed in duplicate using high pH anion exchange chromatography using a DX-500 BioLC chromatograph (Dionex, Sunnyvale, CA) equipped with an AS3500 autosampler (Thermo Separation Products, San Jose, CA) with pulsed amperometric detection.
Characterization of the Oxidase-The apparent subunit molecular weight of the oxidase was determined using 10% SDS-polyacrylamide gels with a 4% stacking gel. Gels were stained with Coomassie Blue and scanned with a Bio-Rad Gel Doc 1000. Molecular weight standards were bovine serum albumin, Escherichia coli ␤-galactosidase, bovine liver glutamate dehydrogenase, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, chicken egg ovalbumin, phosphorylase b, phosphofructokinase, and myosin from rabbit muscle.
Size exclusion chromatography of the native oxidase was performed in 100 mM potassium phosphate buffer, pH 7.5, containing 100 mM KCl at 20°C using a Superose 12 HR 10/30 FPLC column. The apparent native molecular weight was deduced in comparison with the following standards: aldolase, ferritin, medium chain acyl-CoA dehydrogenase, bovine (␥-globulin, chicken ovalbumin, bovine serum albumin, and thyroglobulin. A sample of the purified oxidase was applied to Immobilon-P from Millipore and shown to be a glycoprotein with the periodic acid-Schiff reaction (27).

RESULTS
Purification of the Oxidase-The egg white sulfhydryl oxidase was purified to homogeneity following the yellow color of the flavoprotein because its catalytic identity was initially unknown (see above). However, the large amount of yellow riboflavin-binding protein present in the eggs from normal chickens complicated this approach. Hence, we used egg white from a strain of chickens that lack a functional riboflavin-binding protein and therefore do not accumulate significant riboflavin in their eggs (13,18,19). This greatly simplified the early phase of this project. For consistency, we have continued to use the mutant egg white as a source of sulfhydryl oxidase, although egg white from normal chickens appears to contain comparable sulfhydryl oxidase activity (using dithiothreitol as a substrate; see "Experimental Procedures"; not shown).
The purification procedure is described under "Experimental Procedures." The key step is the initial batch absorption of the oxidase using small amounts of anion exchange cellulose added to a large volume of diluted egg white (see "Experimental Procedures"). This step results in a dramatic initial purification (an approximately 1000-fold decrease in protein; Table I). Difficulties in assaying the oxidase in unfractionated egg white and in the spectrophotometric measurement of flavin absorbance in this turbid solution preclude relevant entries in Table  I. Ammonium sulfate fractionation of the absorbed proteins, followed by hydrophobic interaction and gel filtration steps, yields the pure oxidase. The enzyme is stable for months when stored at Ϫ20°C.
Physical Characterization of the Oxidase-The oxidase exhibits a typical unresolved flavin spectrum with an absorbance maximum at 454 nm and a second peak at 364 nm (Fig. 1). The flavin was released from the protein by denaturation using methanol or boiling (see "Experimental Procedures") and found to be FAD by its fluorescence and chromatographic properties (see "Experimental Procedures"). The extinction coefficient of this noncovalently bound FAD was 12.5 mM Ϫ1 cm Ϫ1 (by the guanidine hydrochloride method; Ref. 28). Egg white sulfhydryl oxidase shows less than 1% of the fluorescence of an equivalent concentration of FAD (pH 7.5, 25°C) exciting at 450 nm.
SDS-polyacrylamide gel electrophoresis (10%) of the pure oxidase revealed a single, rather diffuse, band with an apparent molecular weight of ϳ93,000 (see "Experimental Procedures"). The enzyme was found to be a glycoprotein using the periodate-Schiff reagent (see "Experimental Procedures") and by carbohydrate analysis (see Table II). The minimum molecular weight per FAD was calculated to be 67,000 based on amino acid content and about 80,000 including carbohydrate (Table II). This latter value is consistent with the apparent molecular weight of 93,000 observed on SDS-polyacrylamide gel electrophoresis, since glycoproteins typically run more slowly than expected. Gel filtration of the native sulfhydryl oxidase (see "General Methods") gave an apparent molecular weight of 202,000. These data are consistent with a homodimeric structure for the oxidase with one molecule of FAD per subunit.
The native oxidase does not react significantly with DTNB at pH 7.5 (see "Experimental Procedures"). Similarly, the oxidized enzyme incubated with 1 mM iodoacetamide for 24 h under nondenaturing conditions at pH 7.5 showed only 0.3 molecules of S-carboxymethylcysteine/subunit. When the enzyme was denatured with 5.8 M guanidine hydrochloride before the addition of DTNB, 1.05 thiol residues/subunit were exposed. Essentially the same result (0.9 thiols) was obtained denaturing the enzyme first under anaerobic conditions to ensure that oxidation of free thiols by any enzyme that had yet to be inactivated had not occurred. Exhaustive reductive alkylation (see "Experimental Procedures") gave a total of 5.4 molecules of S-carboxymethylcysteine/subunit. These data are consistent with a subunit containing two disulfide bridges and one free sulfhydryl group that is unreactive in the native oxidized enzyme (see below).
Catalytic Activity-Like the other sulfhydryl oxidases (9 -11) the consumption of oxygen could be conveniently monitored in the oxygen electrode using air-saturated buffers at pH 7.5. The addition of limiting amounts of the dithiol DTT (to a final concentration of 150 M) caused the uptake of 140 M oxygen. Discharge of the hydrogen peroxide upon the addition of catalase led to the reappearance of 72 M oxygen (not shown; see "Experimental Procedures") in agreement with the expected stoichiometry for the sulfhydryl oxidases (2). Table III summarizes the turnover numbers and K m values for a series of common thiol reagent determined at pH 7.5. Despite a wide range of K m values (from 0.15 mM for DTT to 54 mM for ␤-mercaptoethanol), the maximal turnover numbers are rather similar. Further work will determine whether this reflects a common rate-limiting step after the initial reduction of the enzyme by thiol reagents (see below). When assay traces are recorded using a large excess of thiol over dissolved oxygen, e.g. 5 mM GSH or DTT (compared to 0.24 mM oxygen), almost linear rates are obtained for 90% of the trace. The K m for oxygen is thus less than 20 M and cannot be reliably determined with these experiments.
The apparent pH dependence of the oxidation of dithiothreitol by the egg white oxidase is shown in Fig. 2. Deprotonation of a group with an apparent pK of 5.2 leads to an increase in activity. A small loss of activity at high pH with an apparent pK of 9.3 is observed (Fig. 2). Consequently, the oxidase is active over a rather broad pH range (greater than 75% of maximal activity is observed from pH values of 6 -10).
The k cat /K m for oxygen with these thiol substrates is greater than 10 6 M Ϫ1 s Ϫ1 (assuming a K m of less than 20 M), so there appears to be little doubt that oxygen is the physiological electron acceptor of this enzyme. However, we have found that sulfhydryl oxidase, like glucose oxidase (29) and several other flavoenzymes (17,30,31), can utilize the ferricenium ion as an alternate oxidant even in air-saturated solutions. Assays of sulfhydryl oxidase were performed spectrophotometrically at pH 6 (see "Experimental Procedures") to minimize the nonenzymatic oxidation of dithiothreitol by the ferricenium ion.  Amino acid and carbohydrate analysis were performed as described under "Experimental Procedures" and are expressed as residues/FAD. Total cysteine content was that of S-carboxymethylcysteine (SCMC) after denaturation, reduction, and alkylation with iodoacetate (see "Experimental Procedures"). Tryptophan content was estimated spectrophotometrically (Refs. 23 and 24; see "General Methods"). The subunit molecular weight based on the amino acid composition is 67,000, and that including the carbohydrate and FAD content is approximately 80,000. Residues of amino acids or monosaccharide are rounded to the nearest integer.  Fig. 3 shows a dithionite titration of the egg white oxidase under anaerobic conditions at pH 7.5. Complete reduction of the enzyme flavin requires a total of 4.6 electrons (curve 1 to curve 5; see inset). The long wavelength band observed at intermediate dithionite additions (maximal at 2.3 electrons) shows an absorbance maximum at about 560 nm without the fine structure expected for a flavin blue radical (32). Rather, this species resembles the thiolate to flavin charge transfer complexes observed in the flavin disulfide oxidoreductase family (EH 2 , Scheme 1; Ref. 15). Further evidence to support the involvement of a redox-active disulfide in the egg white oxidase will be presented later. The fully reduced oxidase (curve 5) is rapidly reoxidized in the presence of air. Reduction of the enzyme after each aliquot of sodium dithionite was rapid, and the spectral changes after each addition were stable. In addition, sodium borohydride and catalytic photoreduction (33) readily reduced the enzyme with exactly the same progression of changes seen in Fig. 3 (not shown; see "Experimental Procedures"). The fact that all reduction methods (including the use of thiol substrates; see below) gave essentially the same yield of EH 2 suggests a facile equilibration of redox states in the oxidase.
The same long wavelength intermediate is observed during anaerobic titration of the oxidase with thiol reagents. Fig. 4 shows a titration with reduced dithiothreitol, and the inset plots the absorbance changes at 454 and 560 nm with equivalents of DTT added. The long wavelength band generated (maximal at 1.05 mol of DTT/mol of FAD; apparent molar absorp-tivity 2.5 mM Ϫ1 cm Ϫ1 ) appeared at the same position as that generated by sodium dithionite or borohydride. Early spectra in the titration show an isosbestic point at 505 nm, corresponding to the conversion of oxidized enzyme to the EH 2 state before the fully reduced form of the enzyme begins to accumulate. Full reduction required 2.2 equivalents of the two-electron donor, DTT (4.4 electrons; see inset).
Reduced glutathione effects essentially identical spectral changes (not shown), but an approximately 7-fold molar excess is required to reach maximal charge transfer complex formation, and about 100 mol of GSH are required for complete conversion to the EH 4 state (Fig. 5). These differences presumably reflect the fact that GSH is a thermodynamically weaker reductant than DTT (Ϫ0.24 versus Ϫ0.31 V; Ref. 34).
Reactivity of Sulfhydryl Oxidase with Sulfite-Flavoprotein oxidases usually react with sulfite to generate N-5 flavin adducts in a reaction that leads to substantial bleaching of the flavin chromophore at 450 nm (35,36). The egg white oxidase provides an interesting variation to this generalization (Fig. 6). Sulfite addition at pH 7.5 under aerobic conditions leads to the appearance of a long wavelength species very similar, but of weaker intensity, to that encountered in the reductive titrations of Figs. 3-5. Difference spectra show that the position of the long wavelength maximum is essentially the same between reductive and sulfite titrations (not shown). An important difference, however, is that the spectral changes induced by sulfite are stable in air. Thus, the enzyme was held on ice for 72 h after the completion of the titration shown in Fig. 6 without a significant decline in the long wavelength band (not shown). Data from all these spectrophotometric titrations (Figs. [3][4][5][6] are consistent with involvement of a redox-active disulfide in the catalysis by the oxidase (see below).
Further Evidence for a Catalytically Important Redox-active Disulfide-The activity of the egg white oxidase is essentially unchanged after a 20-h incubation with 1 mM iodoacetamide or iodoacetic acid (104 and 103% of control levels, respectively; see "Experimental Procedures"). Further, only about 0.2 thiols react in a 20-min incubation of the native oxidase with 0.5 mM DTNB in phosphate buffer, pH 7.5 (not shown). However, the oxidase became sensitive to alkylation with either iodoacetamide or iodoacetate when reduced with sodium dithionite. Fig.  7 shows a dithionite titration of the oxidase in the presence of 1 mM iodoacetamide. Initial additions of dithionite gave the   2. pH dependence of the activity of sulfhydryl oxidase toward DTT. The activity of egg white sulfhydryl oxidase was measured in the oxygen electrode using 10 mM DTT in air-saturated buffers (see "Experimental Procedures"). The curve is the nonlinear least squares fit with limiting values for the turnover at low, intermediate, and high pH values of 0, 1050, and 740/min respectively and pK values of 5.2 and 9.3. 560-nm-long wavelength band observed previously, but the intermediate decayed to an oxidized flavin spectrum after about 20 min. The reversion to an oxidized flavin spectrum is responsible for the lag phase seen in the inset (Fig. 7). Curve 2 is after the addition of 2.4 electrons. Subsequent reduction of the flavin (curves 3-6) proceeds without the accumulation of significant semiquinone or long wavelength species (Fig. 7). After incubation of the oxidase for 12 h, the enzyme was allowed to reoxidize in air and assayed in the oxygen electrode. The iodoacetamide-treated reduced enzyme showed 2.3% of control activity. Comparable experiments with 1 mM iodoacetate gave analogous spectral changes with a residual activity of 3.5% (data not shown). Alkylation effects very small changes in the visible spectrum of the oxidase (not shown). There is a 1-2-nm blue shift in the main absorbance peak and a slightly increased absorbance below 400 nm for the iodoacetate-modified protein. In contrast, iodoacetamide-treated enzyme shows an almost undetectable blue shift in the 454-nm absorbance envelope. Neither of the carboxymethylated proteins regained significant activity after the removal of excess reagents by ultrafiltration.
Amino acid analysis of the oxidase alkylated with iodoacetamide in the absence of reduction shows essentially no Scarboxymethyl cysteine. Reduction as in Fig. 7 gave approximately 2.7 residues of S-carboxymethylcysteine/subunit of the oxidase. Table IV summarizes properties of flavoprotein sulfhydryl oxidases purified to date. All contain FAD and are either monomeric or dimeric proteins. The enzymes show a range of specificities from those with a pronounced preference for glutathione (e.g. the Penicillium enzyme; Ref. 11) to those active with a comparatively broad range of substrates. All of the oxidases except the Penicillium enzyme significantly accelerate the reoxidation of reduced ribonuclease (Refs. 9 and 10; see below).

DISCUSSION
The data in this paper strongly suggest that the egg white sulfhydryl oxidase contains a redox-active disulfide bridge functionally equivalent to those found in the flavin disulfide SCHEME 1. Some redox and chemically modified states of egg white sulfhydryl oxidase. E, EH 2 , and EH 4 represent oxidized, 2ereduced, and 4e-reduced enzyme without regard to protonation state. Sulfite attacks the interchange thiol, at least in part, leaving the charge transfer thiol free to interact with the oxidized flavin moiety. Alkylation of the reduced enzyme labels both charge transfer and interchange cysteine residues. EH 2 is shown as an equilibrium between dithiol and dihydroflavin states (15). oxidoreductase family. A preliminary report on the sulfhydryl oxidase from Aspergillus niger reached a similar conclusion (37). A body of evidence summarized previously (15) shows that the two cysteine residues in glutathione reductase and lipoamide dehydrogenase, for example, play distinct roles in catalysis. The charge transfer thiolate mediates redox communication with the flavin via formation of a transitory C-4a adduct (15). We arbitrarily depict the lower sulfur atom in Scheme I as reactive with external nucleophiles such as reduced glutathione or sulfite (see below). The upper thiol communicates primarily with the flavin prosthetic group and thus forms the charge transfer complex seen in Fig. 5.
The egg white oxidase gave pronounced long wavelength bands at the midpoint of reductive titrations for all reagents used (e.g. Fig. 3-5). Weak long wavelength bands were observed with both the rat seminal oxidase reduced with excess (10 mM) ␤-mercaptoethanol and the A. niger enzyme treated with 20 mM GSH. Similar substantially bleached flavin spectra with a small residual long wavelength (EH 2 ) component were observed toward the completion of DTT and GSH titrations of the egg white enzyme (not shown). There can be little doubt that all of these enzymes show thiolate to flavin charge transfer complexes at the EH 2 level (Scheme I, dotted lines) by analogy with precedents set earlier (15,38).
The alkylation data of Table II are consistent with the alkyl-ation of both redox-active cysteine residues in EH 2 as observed with thioredoxin reductase (Scheme I; Refs. 15,39). Comparable experiments with lipoamide dehydrogenase, glutathione reductase, and mercuric reductase show monoalkylation of the N-terminal redox-active cysteine residue (40 -42). Presumably in these cases, the charge transfer thiol is unreactive because it is inaccessible to relatively bulky and polar alkylating reagents (15). The behavior of the egg white oxidase toward sulfite is unusual, although sulfitolysis of disulfides is well known (43,44). The weaker intensity of the charge transfer band observed in Fig. 6 compared with those seen with dithionite or DTT might reflect a change in the environment and/or pK of the upper thiol in Scheme I accompanying polar modification of the lower thiol. Alternatively, the reaction with sulfite might occur at either upper or lower thiols (of which only the lower is shown in Scheme I). The failure of lipoamide dehydrogenase and glutathione and thioredoxin reductases to yield comparable adducts (35) might reflect their lower redox potential (and hence a greater resistance of their active site disulfides to sulfitolysis). The A. niger oxidase gives a partial bleaching of the enzyme flavin, consistent with formation of a sulfite adduct of the flavin (10,35). In contrast, the rat seminal enzyme was reported not to react with sulfite (9). The reasons for the differences in behavior between the flavoprotein sulfhydryl oxidases are not yet understood.
The marked similarities in behavior between the sulfhydryl oxidase and the pyridine nucleotide flavin disulfide oxidoreductases could reflect a common evolutionary origin for these enzymes (15). If the oxidase has evolved from this disulfide oxidoreductase family, it has not retained a functional pyridine nucleotide binding site; the enzyme shows no detectable spectral changes when mixed anaerobically with either 100 M NADH or NADPH (see "Experimental Procedures"; data not shown). Similarly, the flavoprotein oxidases from seminal vesicles and Aspergillus fail to communicate with pyridine nucleotides (9, 10). In contrast, a possible convergent solution to the problem of efficient communication between exogenous thiols and flavin might have involved recruitment of a suitably placed disulfide bridge from a preexisting flavoprotein oxidase family of which glucose oxidase and alcohol oxidase are members (45). Divergent and convergent mechanisms might both participate in the evolution of the sulfhydryl oxidases.
A number of reports have suggested a role for sulfhydryl oxidases in protein disulfide bond formation, and an association of the enzyme with tissues active in protein secretion has been noted (2,5,9,10,46). Both the Aspergillus and the seminal enzyme were found to catalyze the reoxidation of reduced ribonuclease at apparently modest rates. The Penicil-  7. Alkylation of the reduced oxidase with iodoacetamide. Sulfhydryl oxidase (7.4 M in 100 mM phosphate buffer, pH 7.5, containing 0.3 mM EDTA; spectrum 1) was mixed with 1 mM iodoacetamide and titrated anaerobically with standardized dithionite (see "Experimental Procedures"). Spectra 2-6 were recorded approximately 20 min after the addition of 1.20, 1.44, 1.68, 1.92, and 2.40 mol of dithionite/mol of flavin (see "Results"). The inset plots the absorbance at 454 nm throughout the titration.
lium oxidase was reported to be unable to catalyze protein disulfide bond formation (11). Preliminary experiments with the egg white oxidase suggest that it effects rapid disulfide bond formation in reduced ribonuclease. 2 Since egg white has an aggregate disulfide concentration of about 10 -15 mM, 3 a role for sulfhydryl oxidase in disulfide bond formation is plausible. Egg white proteins are synthesized directly in the oviduct and it will be interesting to see whether these secretory cells themselves contain high levels of sulfhydryl oxidase activity. It should be noted that several other pathways for the net synthesis of disulfide bridges in proteins have been suggested including a microsomal hydroxylase (47) and a route involving the preferential transport of oxidized glutathione into the endoplasmic reticulum (48). Since egg white is a self-contained package of secreted proteins, it may prove useful in the study of the synthesis and isomerization of protein disulfides.
In conclusion, the egg white oxidase provides a valuable complement to earlier studies on the seminal and microbial sulfhydryl oxidases. Further work will explore the mechanism, structure, and physiological role of these interesting but understudied enzymes.


extfieldsofstudy: Biology Medicine

text:
Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.

The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces, each having a distinct protein and lipid composition. Cells use two pathways to deliver proteins to the correct surface. First, newly made proteins are packaged in the trans-Golgi network (TGN) 1 into vesicles that deliver them directly to either the apical or basolateral surface. Second, proteins can be delivered first to one surface and then endocytosed and transcytosed to the opposite surface. At least in the well polarized Madin-Darby canine kidney (MDCK) cell line, targeting to the basolateral surface generally requires a sorting signal located in the cytoplasmic domain of the protein. For a number of basolateral proteins it has been found that mutations in the cytoplasmic domain prevent TGN to basolateral targeting (1). The first basolateral signal to be identified is the 17-amino acid membrane proximal segment (residues 653-670) of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) (2). This protein is normally delivered from the TGN to the basolateral surface, and then endocytosed and transcytosed to the apical surface. Deletion of most of this 17-residue segment prevents TGN to basolateral delivery. Moreover, this segment can be transplanted to a heterologous reporter molecule, which is then retargeted from the apical to the basolateral surface (2). In at least one other case, the low density lipoprotein receptor, autonomous basolateral sorting signals have been identified (3).
The pIgR basolateral targeting signal has been systematically analyzed by alanine scanning mutagenesis (4). Mutation of His-656, Arg-657, or Val-660 to Ala substantially diminishes, but does not eliminate, TGN to basolateral targeting. Mutation of other residues had little or no effect. The structure of a synthetic 17-residue peptide corresponding to this signal has been determined by two-dimensional nuclear magnetic resonance spectroscopy (4). This peptide tends to adopt a putative type 1 ␤-turn, encompassing residues 658 -661, followed by a nascent helical structure. In MDCK cells polarized sorting takes place in both the TGN and after endocytosis. Mutations that diminish TGN to basolateral sorting and increase TGN to apical sorting have a similar effect on sorting in the endocytotic pathway, decreasing recycling to the basolateral surface and increasing transcytosis to the apical surface (5). These data indicate that the basolateral targeting signal also functions as a signal to retrieve the pIgR from the endocytotic pathway to the basolateral surface. Very similar results have been obtained with the two basolateral sorting signals of the low density lipoprotein receptor (6). This suggests that the same sorting machinery operates in both the TGN and the endocytotic pathway, or that sorting in both pathways actually 1 The abbreviations used in this paper are: TGN, trans-Golgi network; pIgR, polymeric immunoglobulin receptor; CaM, calmodulin; d/pIgA, dimeric/polymeric immunoglobulin A; VSVG, vesicular stomatitis virus protein G; BS 3  occurs in a common compartment.
Transcytosis of pIgR is regulated by several mechanisms. Phosphorylation of Ser664, which is part of the basolateral sorting signal, stimulates transcytosis. Mutation of Ser-664 to Ala (S664A) reduces transcytosis, while mutation to Asp (S664D), whose negative charge may mimic a phosphate, stimulates transcytosis (7). Phosphorylation apparently works by weakening the basolateral signal, since the S664D mutant exhibits decreased TGN to basolateral sorting, increased TGN to apical sorting (5), as well as decreased recycling and increased transcytosis after endocytosis.
Binding of the ligand, dimeric IgA (dIgA) to the pIgR also stimulates transcytosis. Ligand binding stimulates transcytosis of the wild-type pIgR, as well as S664D and S664A (8,9). Ligand-dependent stimulation suggests that the pIgR may transduce a signal to the cytoplasm. Indeed, we have recently found that dIgA binding causes tyrosine phosphorylation of a phosphatidylinositol-specific phospholipase C␥1, activation of protein kinase C, and production of inositol 1,4,5-trisphosphate. 2 Transcytosis of pIgR and other molecules is stimulated either by activation of protein kinase C (10) or by increase in intracellular free Ca 2ϩ , 3 so both arms of the phospholipase C signaling pathway may redundantly stimulate transcytosis. Delivery to the apical surface, either by transcytosis or directly from the TGN, is also stimulated by the heterotrimeric G protein, G s (11,12), as well as cAMP and protein kinase A (13,14). Both the G s ␣ and G␤␥ subunits are stimulatory, at least for transcytotic apical delivery (11). Transcytosis in MDCK cells is stimulated by bradykinin, 3 while in animals transcytosis is stimulated by several hormones and neurotransmitters (15)(16)(17)(18). Presumably, these extracellular signals work through the various second messengers mentioned above.
Transcytosis of the pIgR can be divided by both morphological and biochemical assays into at least three steps. Step 1 is internalization via clathrin-coated pits into basolateral early endosomes.
We are interested in molecules that bind to the cytoplasmic domain of the pIgR, particularly its basolateral sorting signal, as such molecules might be involved in basolateral sorting and/or regulation of traffic. The best evidence for a candidate molecule capable of recognizing and deciphering a basolateral targeting signal is a protein of ϳ220 kDa apparent molecular mass that has been cross-linked to the cytoplasmic domain of the vesicular stomatitis virus G protein (VSVG) (23). A peptide corresponding to the cytoplasmic domain of VSVG prevented cross-linking and also inhibited TGN to basolateral transport of VSVG in permeabilized MDCK cells, while a peptide corresponding to a mutant VSVG that is not basolaterally targeted prevented neither cross-linking nor basolateral transport. However, a peptide corresponding to the pIgR 17-amino acid basolateral targeting signal did not inhibit basolateral transport of VSVG (23), suggesting that this protein might not recognize the pIgR basolateral targeting signal.
Studies on MDCK cells using CaM antagonists suggest that CaM is important in endosome function (24,25). We now report that in the presence of Ca 2ϩ , calmodulin (CaM) binds to the basolateral sorting signal of the pIgR. An analysis of mutant pIgRs reveals that the sequence requirements for basolateral targeting of the pIgR and CaM binding are not identical, suggesting that the role of CaM binding is not to target the pIgR to the basolateral surface, but rather may be involved in regulation of pIgR transcytosis.

MATERIALS AND METHODS
Isolation of Rat Liver Subcellular Fractions-Three rat liver endosomal subfractions, designated early endosomes (CURL, compartment for uncoupling of receptors and ligands), late endosomes (MVB, multivesicular bodies), and a receptor recycling compartment (RRC), were isolated from estradiol-treated rats as described previously (26). Endosomal membranes were separated from contents after rupturing the organelles in a French pressure cell (26).
Antibodies-Unfractionated ascites fluid containing the monoclonal antibody SC166, which recognizes the cytoplasmic domain of the rabbit and rat polymeric immunoglobulin receptor (pIgR), was prepared as described elsewhere (27). A sheep polyclonal antibody made against rabbit secretory component (SC) has also been described (28).
Two-dimensional Electrophoresis and Western Blotting-Two-dimensional electrophoresis was carried out as described (29). After electrophoresis, proteins were transferred to nitrocellulose filters at 20 V overnight at 4°C. Filters were incubated in phosphate-buffered saline containing 5% powdered skim milk to block nonspecific sites. The nitrocellulose was then incubated with a 1/1000 dilution of the monoclonal ascites SC-166 directed against the cytoplasmic tail of the pIgR in phosphate-buffered saline containing 0.05% Tween 20, followed by horseradish peroxidase-conjugated anti-mouse IgG (Jackson Immu-noResearch Laboratories, West Grove, PA), and detection with 4-chloro-1-naphthol as described by Burnette (30).
Calmodulin Overlay Technique-For the 125 I-CaM overlay of twodimensional gels, the method described by Bachs and Carafoli (31) was used. Calmodulin, isolated as described (32) or purchased from Sigma, was radioiodinated with the Bolton-Hunter reagent (Amersham Corp.). Endosome fractions enriched in CURL and RRC show similar protein compositions and give similar results in 125 I-CaM overlays. An experiment using RRC is shown under "Results" (Fig. 1).
MDCK Clones Expressing pIgR Constructs-All nucleotide and amino acid positions are according to the original published rabbit pIgR sequence (33). MDCK strain II cells expressing wild-type pIgR (34), a mutant lacking a membrane-proximal 14-amino acid segment of the cytoplasmic tail required for basolateral targeting (pIgR ⌬655-668) (2), and a "tail-minus" construct lacking all but the two membrane-proximal amino acids of the cytoplasmic tail (2, 35) termed R655-STOP (4), have been described previously. An MDCK clone expressing pIgR in which the cytoplasmic domain consists only of the membrane proximal 17-amino acid basolateral targeting signal (T670-STOP) (4), and point mutants in which individual residues in the pIgR 17-amino acid basolateral targeting signal are replaced with alanine in the context of the entire cytoplasmic tail (H656A, R657A, N659A, and V660A) (5) have also been described. These mutants are indicated by the wild-type amino acid, followed by the position and the new amino acid (e.g. H656A designates a mutant where histidine at position 656 has been mutated to alanine). A construct encoding the full-length pIgR molecule with a substitution of alanine for arginine at position 658 was constructed by ligating a DNA fragment encoding the C terminus (residues 661-755) of wild-type pIgR clone to a fragment encoding residues 1-660 derived from the R658A/T670-STOP mutant described previously (4) via a common AvaII site at nucleotide position 2007. The resulting cDNA was ligated into the BglII site of pCB6 (36) and transfected into MDCK strain II cells as described previously (4). Mutants in which serine 664 has been mutated to alanine (S664A) or aspartic acid (S664D) have also been described (7).
Immunoprecipitation-Immunoprecipitation of pIgR was performed using protein G-Sepharose (Sigma) coupled to polyclonal sheep anti-SC IgG, essentially as described (4).
Calmodulin Affinity Chromatography-For CaM-agarose binding assays, MDCK cells expressing pIgR constructs were grown on 24-mm Transwell filters (0.4-m pore size, Costar Corp., Cambridge, MA) metabolically labeled at 37°C for 40 min on an 80-l drop of Dulbecco's modified Eagle's medium lacking cysteine (obtained from the UCSF cell culture facility, supplemented with 5% dialyzed fetal bovine serum) containing 25 Ci of [ 35 S]cysteine (DuPont NEN). The Transwell filters were washed three times with phosphate-buffered saline containing CaCl 2 and Mg 2ϩ , and filters were cut out and placed in 1 ml of ice-cold solubilization buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 containing 1% Triton X-114 (Boehringer Mann-heim) and protease inhibitors (5 g/ml pepstatin, 10 g/ml chymostatin, 5 g/ml leupeptin, 10 g/ml antipain, 500 M benzamidine, and 0.1% Trasylol) and rotated for 15 min at 4°C. Insoluble material was removed by centrifugation in a microcentrifuge for 15 min at 4°C. The supernatant was transferred to a new tube containing 6 l of 0.5 M EGTA (3 mM final). Triton X-114 phase-partitioning was performed by warming to 37°C for 3 min, followed by centrifugation at full speed for 5 min in a microcentrifuge at room temperature. The aqueous phase was discarded, and the detergent phase was resuspended with nine volumes (approximately 0.9 ml) of cold wash buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 3 mM EGTA) and incubated at 4°C for 5 min. The phase-separation procedure was repeated, followed by dilution of the detergent phase with nine volumes of ice-cold binding buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 ). After 5 min at 4°C, the samples were precleared with 50 l of Sepharose CL-2B at 4°C (34).
The sample (500 l) was combined with 450 l of binding buffer and 50 l of a 20% slurry of CaM-agarose, which had been equilibrated with binding buffer. Samples were incubated for 1 h at 4°C on a rotator. After pelleting the CaM-agarose beads briefly in a cold microcentrifuge and aspirating the liquid, the beads were washed once with 1 ml of binding buffer containing 0.5% Triton X-100 and once with binding buffer containing 0.05% Triton X-100. At this point, a 5-min incubation was carried out at 4°C with binding buffer containing 0.05% Triton X-100, with or without 3 mM EGTA. The liquid was separated from the beads as before, and the beads were washed once with binding buffer with or without EGTA (no detergent). The beads (10 l packed) were boiled in 20 l of sample buffer, and half of the sample (ϳ10 l) was analyzed by SDS-PAGE and fluorography or phosphorimaging. Total pIgR in the starting material for CaM-agarose was determine by immunoprecipitation of a duplicate sample. Starting material, typically 500 l, was diluted with an equal volume of 2.5% Triton dilution buffer (2.5% Triton X-100, 100 mM triethanolamine-HCl, pH 8.6, 100 mM NaCl, 5 mM EGTA, 1% Trasylol, 0.02% NaN 3 ) and analyzed by pIgR immunoprecipitation, followed by SDS-PAGE and fluorography or phosphorimaging.
To correct for differences in pIgR concentrations in the starting material due to differing expression levels among clones or different partitioning behavior of constructs in Triton X-114, unlabeled preparations were analyzed by Western blotting to determine the relative concentration of each construct in the preparations. For clones that yield more pIgR in the starting material, the volume of Triton X-114 preparation added was reduced appropriately so that approximately equal amounts of each pIgR (wild-type or mutant) were added to CaM beads. The total amount of Triton-X114 starting material added was kept at 500 l by adding the appropriate volume of an identical preparation from non-transfected MDCK cells.
Cross-linking of CaM to Solubilized pIgR-Triton X-114 preparations (made as described above, except that Hepes was substituted for Tris) of metabolically labeled proteins from MDCK cells expressing pIgR constructs were incubated in the presence or absence of 1 M exogenous CaM, with or without 3 mM EGTA. After 1 h of incubation at 4°C, 0.5 mM BS 3 (Pierce) was added to the samples indicated. Incubation was continued for an additional 1 h, at which time the cross-linker was quenched by addition of glycine to 100 mM. SDS was added to a final concentration of 1%, samples were boiled for 5 min, diluted with an equal volume of 5% Triton dilution buffer (5% Triton X-100, 100 mM triethanolamine-HCl, pH 8.6, 100 mM NaCl, 5 mM EGTA, 1% Trasylol, 0.02% NaN 3 ), precleared once with 50 l of Sepharose CL-2B, and immunoprecipitated for pIgR. Precipitates were washed and analyzed by SDS-PAGE and fluorography.
Cross-linking of CaM to Crude Membranes-MDCK cells expressing pIgR constructs were grown on 15-cm dishes for 3-5 days until confluent. Cells were harvested by scraping in phosphate-buffered saline, pelleted at 150 ϫ g for 5 min at 4°C, washed once in homogenization buffer (50 mM Hepes, pH 7.4, 250 mM sucrose), and resuspended in 2 ml of homogenization buffer containing protease inhibitors by pipetting with a 1-ml micropipette tip. Cells were homogenized by 12 passes through a 22-gauge needle. A post-nuclear supernatant was prepared by centrifugation at 1400 ϫ g for 15 min at 4°C. EGTA was added to the post-nuclear supernatant to 3 mM, followed by centrifugation at 250,000 ϫ g for 1 h at 4°C (68,000 rpm in a Sorvall RP100-AT4). The pellet, containing crude membranes, was resuspended in 50 mM Hepes, 3 mM EGTA. Glycerol was added to 5%, and the membranes were frozen in liquid nitrogen and stored at Ϫ80°C. Just prior to use, samples were thawed and centrifuged at 250,000 ϫ g for 1 h at 4°C, and pellets were resuspended in HNMC (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 ). 125 I-CaM (prepared by the iodine monochloride method; Ref. 37), was incubated with crude membranes in HNMC for 1 h at 4°C in the presence or absence of 3 mM EGTA. BS 3 was added to a final concentration of 0.5 mM, and the incubation was continued for 1 h. Samples were quenched with glycine, boiled in SDS, and immunoprecipitated and analyzed as above.

Identification of a Rat Liver Endosome CaM-binding Protein
as pIgR-CaM has been suggested to play several possible roles in membrane traffic in mammalian cells. We and others have examined the effects of inhibitors of CaM, such as W13 and W7, on various steps in membrane traffic (24,25). In MDCK cells expressing pIgR, these drugs inhibited transcytosis of pIgR and recycling of transferrin, and caused all material endocytosed from both surfaces of the cell to be delivered to exceptionally large, abnormal endosomal structures (25), suggesting a role for CaM in polarized sorting in early endosomes. However, the mechanism(s) by which CaM regulates membrane traffic events are poorly understood.
In order to address the mechanism(s) by which CaM regulates endosomal function, we were interested in identifying major CaM-binding proteins in endosomes. Previous studies showed a major CaM-binding protein of approximately 115 kDa could be demonstrated by 125 I-CaM overlay of two-dimensional protein blots of an endosome-rich fraction from rat liver (38). We discovered that this protein can also be labeled by staining with a monoclonal antibody (SC166) against the polymeric immunoglobulin receptor (pIgR) (Fig. 1). Similar results were obtained with a rabbit antiserum raised against rat SC (data not shown).
Characterization of CaM Binding to pIgR-In order to further support the identification of pIgR as the CaM-binding protein, and to map and characterize the CaM binding site on pIgR, we developed a CaM-binding assay as described under "Materials and Methods." MDCK cells expressing the pIgR were metabolically labeled and solubilized with Triton X-114. Hydrophobic, integral membrane proteins partition into the detergent phase during Triton X-114 phase separation induced by warming to 37°C. Surprisingly, when such a preparation is added to CaM-agarose in the presence of Ca 2ϩ , followed by washing to remove nonspecific binding, pIgR is the major protein detected after the CaM beads are eluted with SDS and subjected to SDS-PAGE and fluorography ( Fig. 2A). This result is striking when one considers that pIgR is only a minor protein in the starting material ( Fig. 2A), and that there are numerous CaM-binding proteins that might be present in the Triton X-114-partitioned material. It is similarly striking that pIgR is the major CaM-binding protein detected in the rat liver membrane fraction (Fig. 1).
Binding of pIgR to CaM-agarose is inhibited by addition of EGTA ( Fig. 2A), excess CaM (Fig. 2B), or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; data not shown), further supporting the specificity of binding. If CaM binding to pIgR is physiologic, then we would expect the CaM binding site to be on the cytoplasmic domain of pIgR. To test this, we used a pIgR construct encoding the extracellular and transmembrane domains of pIgR, but lacking virtually all of the cytoplasmic tail (pIgR R655-STOP). As shown in Fig. 3, this "tail-minus" construct shows no detectable binding to CaM. We used other mutants to localize the site of CaM binding on the pIgR cytoplasmic tail (Fig. 3). Remarkably, a mutant lacking the membrane-proximal 14-amino acid segment of the cytoplasmic tail required for basolateral targeting (⌬655-668) also fails to bind CaM. Conversely, a mutant in which the cytoplasmic domain consists only of the membrane-proximal 17-amino acid basolateral targeting signal (pIgR T670-STOP) exhibits CaM binding indistinguishable from that of full-length pIgR.
Chemical Cross-linking of CaM to pIgR-CaM binding to pIgR was also demonstrated using chemical cross-linking. Metabolically labeled, Triton X-114-solubilized and partitioned material was incubated with the non-reversible cross-linker, BS 3 , in the presence of CaM. Samples were boiled in SDS, and pIgR was immunoprecipitated and analyzed by SDS-PAGE. As shown in Fig. 4, a significant fraction of wild-type pIgR is converted to a slower migrating species. This species is not observed if CaM, cross-linker, or free Ca 2ϩ are omitted from the assay. Furthermore, the apparent M r of the cross-linked complex is consistent with that expected for a stoichiometric pIgR-CaM complex (ϳ125 kDa). Specificity of the cross-linking as a CaM binding assay is further supported by the fact that mutant pIgRs lacking either the entire cytoplasmic tail (pIgR R655-STOP) or 14 amino acids of the basolateral targeting signal (⌬655-668) could not be cross-linked to CaM (Fig. 4). These results suggest that pIgR binds directly to CaM and that it can bind to soluble CaM as well as CaM-agarose.
In order to determine if CaM can bind to pIgR that is still embedded in the membrane, we performed cross-linking experiments using crude MDCK membranes containing unlabeled pIgR. Radioiodinated CaM is incorporated into a complex of 125 kDa in a Ca 2ϩ -dependent manner with membranes containing wild-type pIgR, but not with pIgR ⌬655-668 (Fig. 5) or cells not expressing pIgR (data not shown). These results attest to the likelihood of physiologic binding of pIgR to CaM in intact cells.
CaM Binding to pIgR Point Mutants Does Not Correlate with Their Basolateral Targeting Phenotype-CaM binding to the basolateral targeting signal suggested that it might play a role in pIgR targeting. In order to determine whether there is a correlation between CaM binding and basolateral targeting, we performed CaM-binding assays on many of our pIgR clones that contain alanine point mutations in the basolateral targeting signal. Typical data from these assays are shown in Fig. 6. One mutant that is not basolaterally targeted shows reduced binding to CaM (R657A), but two others that are not basolat- erally targeted (H656A and V660A) exhibit CaM binding that is comparable to wild-type pIgR. R658A, which is basolaterally targeted as accurately as the wild-type pIgR, does not bind well to CaM, while another mutant that is basolaterally targeted (N659A) does bind CaM. Thus, CaM binding does not correlate with the basolateral targeting phenotype of pIgR mutants.
CaM binding of several proteins is known to be modulated by phosphorylation (39). The pIgR has a major phosphorylation site at Ser-664 in the basolateral targeting signal, and phosphorylation of this residue stimulates transcytosis of pIgR (7,8), presumably by inactivating the basolateral targeting signal (5). We therefore assayed the ability of a non-phosphorylatable mutant, S664A, and a mutant that mimics the presence of a phosphate residue, S664D, to bind to CaM. Both of these mutants bind to CaM as well as wild-type pIgR (data not shown).
Thus phosphorylation of Ser-664 probably does not alter CaM binding.

DISCUSSION
CaM binds to and regulates the function of a wide variety of proteins. In most cases this binding occurs only when CaM is "activated" after binding Ca 2ϩ . Because the affinity of CaM for Ca 2ϩ (ϳ10 Ϫ6 M) is above the resting [Ca 2ϩ ] i (ϳ10 Ϫ7 M), CaM is a sensor for transient increases in [Ca 2ϩ ] i , binding to target proteins only when [Ca 2ϩ ] i is elevated (39). CaM has been suggested to play several possible roles in membrane traffic in mammalian cells. Endocytosis in neurons is dependent on the phosphorylation state of dynamin (40), which is in turn a substrate for calcineurin (phosphatase 2B) (41), a phosphatase that is regulated by CaM binding. Cyclosporin A, an immune FIG. 3. CaM binds to the 17-amino acid basolateral targeting signal of pIgR. CaM binding of pIgR constructs was analyzed as in Fig. 2A. Constructs indicated are as follows: wild-type, wildtype pIgR; pIgR ⌬655-668, a construct in which 14 amino acids of the 17-amino acid basolateral targeting signal have been deleted; pIgR R655-STOP, a construct which contains only the first two amino acids of the cytoplasmic tail; pIgR T670-STOP, a construct which lacks all but the 17-amino acid basolateral targeting segment of the cytoplasmic tail. No binding was detected for pIgR ⌬655-668 or pIgR R655-STOP. The cytoplasmic domain of the protein encoded by each construct is indicated schematically at the right.
FIG. 4. CaM can be chemically cross-linked to detergent-solubilized pIgR. Triton X-114 preparations of metabolically labeled proteins from MDCK cells expressing pIgR constructs (see "Materials and Methods") were incubated in the presence or absence of 1 M exogenous CaM, with or without 3 mM EGTA as indicated, at 4°C for 1 h, followed by incubation for another hour in the presence or absence of BS 3 as indicated. Samples were quenched with 100 mM glycine and boiled in SDS, and pIgR was immunoprecipitated and analyzed by SDS-PAGE. With wild-type pIgR, an additional band of ϳ125 kDa (arrow), which represents pIgR cross-linked to CaM, appears only in the presence of CaM, BS 3 , and free Ca 2ϩ . This treatment does not produce an additional band for pIgR ⌬655-668 or pIgR R655-STOP.
FIG. 5. CaM can be chemically cross-linked to membrane-associated pIgR. Crude membrane preparations from MDCK cells expressing pIgR constructs were incubated with 125 I-CaM in the presence or absence of EGTA. After 1 h at 4°C, BS 3 was added to 0.5 mM, incubation was continued for another hour, glycine and SDS were added to 100 mM and 1%, respectively, samples were boiled for 5 min, and pIgR was immunoprecipitated and analyzed by SDS-PAGE and fluorography. 125 I-CaM was incorporated into a ϳ125-kDa species in the presence of membranes containing pIgR, but not membranes from MDCK cells expressing pIgR ⌬655-668. suppressant that functions by binding calcineurin, inhibits Ca 2ϩ /CaM-dependent secretion from pancreatic acinar cells, thereby implicating calmodulin/calcineurin in regulation of this secretion (42). More generally, [Ca 2ϩ ] i is clearly important in many intracellular membrane traffic events, including both regulated exocytosis of granules (43) and synaptic vesicles (44), as well as classically "constitutive" processes, such as endoplasmic reticulum to Golgi transport (45) and nuclear envelope fusion (46) (at least in in vitro systems). It is not known if [Ca 2ϩ ] i acts on CaM and/or some other target in these events.
We have found that CaM binds to the basolateral targeting signal of the pIgR, making CaM the first identified protein shown to bind specifically to a basolateral sorting signal. Binding is strictly dependent on Ca 2ϩ . The pIgR is the major CaMbinding protein detected in membranes of a highly purified rat liver endosome fraction, and is also the major CaM-binding protein detected in a Triton X-114 detergent phase extract from total metabolically labeled MDCK cells that are transfected with pIgR. These striking results suggest that the interaction of CaM and pIgR is of high specificity. We also detected the CaM-pIgR interaction by cross-linking, both in detergent extracts and, more significantly, in non-solubilized crude membranes prepared from MDCK cells. That CaM can bind to pIgR that is still in its native membrane makes it quite likely that under the appropriate in vivo conditions of elevated [Ca 2ϩ ] i , CaM should bind to pIgR. Capturing the interaction of CaM with pIgR in intact cells may be quite difficult, as elevations of [Ca 2ϩ ] i are generally very transient and spatially localized (47).
Using a series of mutant pIgRs expressed in MDCK cells, we mapped the CaM binding site to the membrane-proximal 17 amino acids of the cytoplasmic domain of the pIgR. This same segment has previously been shown to be necessary and sufficient to direct the pIgR to the basolateral surface from either the TGN or the endocytotic pathway (2). However, an analysis of Ala point mutants in this segment indicates that there is not a precise correlation between the residues needed for basolateral sorting and those needed for CaM binding. The sequence of this 17-residue segment is generally comparable with other known CaM binding sites. CaM often binds to a sequence with a preponderance of basic residues near the N terminus, and hydrophobic residues more concentrated near the C terminus (39). This is largely true of this 17-residue segment of the pIgR. It is remarkable that this short region of the cytoplasmic domain of the pIgR therefore has at least two separable functions, basolateral targeting and CaM binding. This segment also has a third function, as a rather weak signal for endocytosis, centered around tyrosine 668 (48). Based on our knowledge of how sorting occurs in other systems, it seems likely that a specific protein complex recognizes the basolateral sorting signal. This complex would be functionally and perhaps structurally homologous to the adaptor proteins found in clathrin-coated vesicles, and to the coatomer involved in traffic in the early portion of the biosynthetic pathway. A likely candidate for a component of the coat involved in basolateral sorting is the p200 protein found in the TGN (49). Such complexes presumably recognize specific signals on the cytoplasmic domains of integral membrane proteins. In the case of the AP2/HA2 adaptor found in the plasma membrane, the signal contains a type 1 ␤-turn (50 -53), perhaps somewhat similar to the type 1 ␤-turn that is an essential part of the basolateral signal of the pIgR (4).
In contrast, CaM generally does not function as an adaptor, i.e. it does not recognize a feature on one protein, and then bind to another protein. Rather, when CaM binds to a site on a protein, a common consequence is that it simply masks that site, preventing it from interacting with anything else (39). We speculate that one possible role of CaM is to sequester the basolateral signal on the pIgR. In the absence of elevated [Ca 2ϩ ] i , CaM would not bind to the pIgR. The hypothetical complex that recognizes the basolateral signal would bind to the signal and direct the pIgR to the basolateral surface. However, when [Ca 2ϩ ] i is elevated, perhaps due to the binding of dIgA to the pIgR or to another hormone signaling event, CaM would bind to pIgR and thereby prevent the hypothetical complex from binding. By masking the basolateral signal, the pIgR would be allowed to be transcytosed to the apical surface.
In pancreatic epithelial cells, the elevation of [Ca 2ϩ ] i in response to extracellular signals is greatest in the most apical region of the cell (54). With strong stimulation a wave of elevated [Ca 2ϩ ] i can then propagate through the cell. It seems likely that in MDCK cells elevation of [Ca 2ϩ ] i would similarly be concentrated in the most apical region of the cytoplasm. As described in the Introduction, the last known stage of pIgR transcytosis (i.e. step 3) is movement from the apical recycling compartment (located immediately beneath the apical plasma membrane) to the apical surface. It is possible that CaM binds primarily to the pIgR in this compartment and is involved in stimulating this step of transcytosis. Consistent with this hypothesis, we have observed that this is the step of transcytosis that is stimulated by dIgA binding (22), as well as by artificially raising [Ca 2ϩ ] i . 3 This localized increase in [Ca 2ϩ ] i could avoid the binding of CaM to pIgR in other locations, e.g. the TGN, which might lead to inappropriate delivery of pIgR to the apical surface. Exocytosis of synaptic vesicles at nerve terminals represents an extreme example of how a highly localized increase in [Ca 2ϩ ] i leads to membrane traffic at a precise location.
Signaling by dIgA binding to the pIgR or other extracellular FIG. 6. CaM binding to pIgR point mutants does not correlate with their basolateral targeting phenotype. CaM-agarose binding of pIgR mutants, in which alanine is substituted at the indicated positions in the context of fulllength pIgR, was analyzed as for Fig. 2A.
Lanes showing an immunoprecipitation of pIgR from the starting material (IP), and 35 S proteins bound to CaM-agarose in the presence of Ca 2ϩ or EGTA are indicated. The percentage of each construct bound to CaM-agarose, relative to wildtype, and basolateral targeting phenotype (*, determined previously; Ref. 4) of each mutant: (ϩ) normal basolateral targeting; (Ϫ), disrupted basolateral targeting, are indicated at right. Values represent the mean of two experiments (see "Materials and Methods" for details). signals leads to activation of several second messenger pathways, which may redundantly stimulate transcytosis. In particular artificial elevation of [Ca 2ϩ ] i stimulates apical transcytosis of several molecules (e.g. transferrin), although these effects are in general smaller than those observed with pIgR. 3 The molecular mechanisms of these effects are unknown. We suggest that the specific binding of CaM to pIgR provides an additional regulatory mechanism, which amplifies the stimulation of transcytosis of pIgR.
CaM binding to the pIgR could have another, non-mutually exclusive role in regulation of pIgR traffic. When dIgA binds to pIgR, the pIgR initiates a signaling cascade involving tyrosine phosphorylation. 2 Preliminary data indicate that mutations in the 17-residue basolateral targeting signal of the pIgR prevent this signaling, suggesting that this segment has an additional role in signal transduction. Perhaps CaM binding to the pIgR serves to sequester this segment and thereby is part of a negative feedback loop that shuts off signaling by the pIgR.
Taken together our data indicate that the CaM binds to a portion of the pIgR that has multiple functions in regulation of polarized traffic. It will be interesting to learn if other receptors bind to CaM and if this is involved in regulation of their traffic by [Ca 2ϩ ] i .