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Helix-helix interactions inside lipid bilayers

Far from being simple hydrophobic anchors, it is now clear that the transmembrane α-helices of integral membrane proteins can participate in strong, specific interactions that are important in their folding and oligomerization. Crystallographic studies of 21 such helices have indicated that these interactions are similar to those described for soluble proteins. Helix-helix interactions are also important in the oligomerization of a number of proteins that have a single transmembrane α-helix. The interactions are rather specific, involving interhelical salt bridges, hydrogen bonds or precise packing interactions. In some cases, such oligomerization is required for exit from the endoplasmic reticulum. The transmembrane helices of some Golgi-residing proteins also contain sufficient information to ensure their retention in this compartment. Finally, interactions between transmembrane α-helices may be important in the mechanism of transmembrane signalling by a number of membrane-bound receptors.


Introduction
While the general notion that membrane proteins often contain largely hydrophobic transmembrane helices has been accepted for more than a decade, the specificity of their mutual interactions and the diversity of their roles are only now becoming apparent. Far from being simple hydrophobic anchors which locate proteins at a membrane, or weakly interacting structural elements which are stabilized by interactions outside the bilayer, it is now clear that transmembrane helices can participate in highly specific interactions. These interactions involve sutficient energy to drive folding or oligomerization in some cases, and are being shown to contribute to an increasingly diverse set of functional roles. In this review, we highlight work of the past year in light of the conceptual and experimental background that went before. A great deal of progress has been made and the pace is quickening. A useful conceptual framework for the consideration of helix-helix interactions within lipid bilayers is provided by the two-stage model for the thermodynamics of folding of integral membrane proteins [1]. In stage I, independently stable 0t-helices are established across the lipid bilayer, and in stage II these interact to form functional transmembrane structures. Thus, the energetics of helix formation can be separated conceptually from those of the interactions between these helices to form higherorder structures. The model can be applied equally well to the helix-helix interactions occurring within polytopic membrane proteins and to those involved in oligomerization.

Structural studies have characterized 21 transmembrane 0c-helices
Most data concerning the structure of a-helical integral membrane proteins comes from X-ray crystallography of bacterial photosynthetic reaction centers (RCs; for reviews, see [2,3]), electron crystallographic [4] and neutron diffraction [5] studies of the structure of bacteriorhodopsin, and, most recently, electron crystallography of the plant light-harvesting complex [6°°], which together have a total of 21 transmembrane helices. The detailed consideration of helix-helix interactions in these cases is complicated by the influences of the abundant cofactors. Nonetheless, a view emerges from these structures that the packing of the interior of integral membrane proteins is as efficient as that generally observed for water-soluble proteins. It is noteworthy that no interhelical salt bridges are seen, in contrast with what is proposed below for interactions between transmembrane a-helices in the oligomerization of some bitopic membrane proteins. Furthermore, each helix contains, on average, less than one interhelical hydrogen bond. In the case of bacteriorhodopsin, for example, the map obtained from electron crystallographic studies [4] suggests that Asp212 in helix G is involved in hydrogen bonding to Tyr57 of helix B, Trp86 of helix C and to Tyr185 of helix F. Residues buried in the interior of these proteins are, on average, more hydrophilic than those which are lipidexposed, and are also found to be the most well conserved between species. From the analysis of Rees et al. [7], this appears to be a general phenomenon for polytopic a-helical integral membrane proteins (e.g. [8]).
In the 6h structure of the trimeric plant light-harvesting complex [6",], two of the three transmembrane a-helices are longer than those seen in bacteriorhodopsin or the RC, and are associated as in a right-handed supercoil [9], separated from one another by a contact distance of 10k. The subunit boundaries in the trimeric complex are not unambiguous, but it is clear that both helix-helix and helix-chromophore contacts stabilize the structure of the individual subunits as well as the interactions between them.
One aspect of the interaction between a-helices in lipid bilayers that has received attention during the past year is the role of proline residues, which occur frequently in the transmembrane domains of transport proteins. A proline residue in the middle of a transmembrane a-helix results, in many (but not all) cases, in a kink in that helix. Analysis of such kinked helices in the RC and bacteriorhodopsin structures has suggested that they tend to bury their convex sides against other helices [10"]. The exception is helix C of bacteriorhodopsin, for which the convex, most hydrophobic, face is in contact with lipid. Consideration of a number of proline-containing transmembrane a-helices predicted from sequence analysis has suggested that, in general, the postulated convex side is the most polar face of the helix, which is predicted to be disposed towards the protein interior or towards the pore of a channel [10o,11.]. Indeed, where charged residues appear in such transmembrane domains, they tend to lie on the expected convex face. In contrast, in water-soluble proteins, the convex side of proline-kinked a-helices tends to be exposed to the solvent. It has also been proposed that proline residues in transmembrane a-helices may provide structural figidi W which optimizes the positioning of important side chains (for a review, see [12]).

Helix-containing fragments of membrane proteins can reassociate
Chymotryptic cleavage of bacteriorhodopsin yields two fragments: C1, which contains five of the seven transmembrane a-helices; and C2, which contains the remaining two helices. The two fragments have been isolated and reconstituted into separate populations of lipid vesicles, and were seen by circular dichroism (CD) to retain their m-helical structure. Upon mixing of the two populations of vesicles, followed by vesicle fusion, the fragments reassociate. The resulting complex binds retinal and acquires spectroscopic properties characteristic of native bacteriorhodopsin, as well as the lattice formation characteristic of purple membrane [13,14] February 1991). These data demonstrate that the covalent linkages between helices A and B and between helices B and C are not required for the correct folding of bacteriorhodopsin, and ~ow that interactions between transbilayer a-helices are a major determinant in the folding of this integral membrane protein.
Similar experiments have also been reported for /ac permease from Escherichia coli, which has 12 putative transmembrane a-helices. Co-expression of a two-helix fragment with a 10-helix fragment [16] or of two sixhelix fragments [17] resulted in restoration of lactose transport, where none of the individual fragments was capable of transporting lactose. It should, however, be noted that the individual fragments appear to be degraded when expressed alone. Functional ]32 adrenergic receptor has also been obtained in Xenopus oocytes by the co-expression of two fragments corresponding to helices 1-5 and helices 6-7, respectively [18]. These results provide further support for the two-stage model for folding of integral membrane proteins [1].

Helix-helix interactions in oligomerization
There has been much study over the past year of the role of intramembraneous helix-helix interactions in the assembly of oligomeric complexes of proteins containing single transmembrane ¢~-helices. In the absence of oligomerization, degradation of subunits can occur in the endoplasmic reticulum (ER). The main system for such studies is that of the T-cell receptor (TCR4 for a review, see [19]). It has been shown that the single transmembrane domain of TCRa, which includes two basic amino acid side chains, can target this protein for degradation in the ER (for a review, see [20]). Furthermore, a nineamino-acid segment from the transmembrane domain of TCR~, which includes these two basic residues, contains all of the information necessary for its association with CD38, which has one acidic residue in its single transmembrane domain [21]. The interaction of TCRa with CD38 masks determinants in both of these proteins for ER degradation, i.e. in the absence of CD36, TCR0t is degraded, whereas in the absence of TCRa, CD38 is degraded. Thus, it appears that the transmembrane determinants for association of TCRa with CD38 and for ER degradation are co-localized [22], such that if oligomer* ization occurs, degradation is blocked.
Reports published this year have delineated further the roles of the potentially charged residues in the transmembrane domains of these receptor subunits. Bonifacino et a/. [23"] reported that a single arginine or aspartic acid residue can cause targeting for retention and degradation in the ER when placed at central positions within the transmembrane domain of the Tac antigen, which is normally transported to the cell surface. One hypothesis that arises from these data is that interaction of these polar groups with a transmembrane domain of a protein of the ER degradation apparatus may occur. The same laboratory also reported further studies concerning the role of these potentially charged residues in the assembly of the TCR complex [24..]. In a chimeric protein consisting of the extramembraneous portion of Tac and the trans-membrane domain of TCR0t, mutation of either one of the basic residues has relatively little effect upon interaction with CD38, whereas mutation of both of them completely abrogates the association. The TCR clonotypic 8 chain, which has the same two basic residues in its transmembrane domain as TCR~t, was also able to interact with CD38. Mutation of one residue in the transmembrane domain of Tac to an arginine could cause this protein to interact with CD38 in much the same way as such mutations cause Tac to be degraded in the ER [23°°]. Mutation of the single aspartic acid residue in the transmembrane domain of CD38 abrogated the interactions of this protein with TCRa and TCR8 as well as the Tac mutants bearing a basic residue in the transmembrane domain. By altering the position of this aspartic acid residue within the transmembrane domain, the influence of the position of the arginine residue within transmembrane domain of Tac upon its association with CD38 was altered. Most effective interaction was seen when the acidic residue of CD38 and the basic residue introduced into Tac were such that these residues would be at approximately the same level in the lipid bilayer. These results were interpreted as suggesting that interhelical salt bridges, which are expected to be very strong in the low dielectric environment of the membrane [25][26][27], are important in the assembly of the TCR complex. It should be noted that the intramembraneous interaction may either occur between formal charges or involve strong hydrogen bonding of the uncharged groups [25][26][27]. In contrast with this case, no interhdical salt bridges are seen in the RC structures determined at high resolution, or in bacteriorhodopsin.
A situation similar to that for TCR assembly is also seen for the assembly of the transmembrane form of the Fc~/ receptor (Fc~/RIII or CD16) (for reviews, see [28,29]). The amino acid sequence of the single transmembrane domain of this receptor is highly conserved between species, as is also true for other Fc~/Rs [28]. In order for the 0t-subunit of Fc),RIII to reach the plasma membrane, it must associate with either the ~/-subunit of FceRI or the 4-subunit of the TCR/CD3 complex or both (as a3'2, 0~42 or a)'4) [30"]. The ~/-and 4-subunits both have a single transmembrane domain, which is almost identical in the two cases, and contains one aspartic acid residue. I~ a manner analogous to that described for the TCR0~-CD38case, ), or 4 protects Fc~,RIII~t from degradation in the ER. Studies of chimeric proteins [30"] suggest, as for TCRa, that the signal that determines the degradation of Fc),RIRat in the ER resides in its transmembrane domain, which contains an aspartic acid residue. Interaction between the transmembrane domain of the a-subunit and that of ~/or 4 appears to mask this determinant. This hypothesis is strengthened by the finding that substitution of an isoleucine for a leucine residue in the transmembrane domain of human 4 reduces the extent of its interaction with Fc~/RIIIa by 65 %. This leucine is conserved in human 7, but is an isoleucine in mouse 4. Mutation of this isoleucine in mouse 4 to leucine led to a fivefold increase in its association with Fc),RIIIa. No such effects were seen when mutations were made at the few other positions not conserved in mouse 4. Thus, these data clearly suggest that specific interactions between transmembrane a-helices are important ih this assembly process. Each of the aspartic acid residues in the transmembrane domains of a, 4 and "f is important for the interhelical interactions.
There exist other examples of ER retention signals in transmembrane domains which do not involve potentialy charged residues. For example, membrane IgM is retained in the ER of non-B cels. Its transmembrane domain contains a number of wel conserved amino acids with hydroxyl side chains, the mutation of which to aliphatic residues abrogates this ER retention [31]. Some of these residues are also important in the mechanism of transmembrane signalling of IgM in B cells [32].
In addition to these studies of ER retention and degradation signals within transmembrane domains, there have also been reports over the past year of Golgi retention signals in the transmembrane domains of a number of proteins. Through the construction of a series of chimeric molecules and analysis by immunofluorescence microscopy, it has been shown that the transmembrane domain of N-glucosaminyltransferase I is sufficient to confer Golgi retention on several heterologous proteins [33°°]. Similar results have been obtained for the transmembrane domains of a2,6-sialyltransferase (ST) [34"] and [31,4-galactosyltransferase (GT) [35",36°°,37°']. In the latter case, a 10-amino-acid region from the lumenal half of the transmembrane domain was sufficient for retention of a heterologous protein in the trans Golgi cistemae [36"]. That the lumenal half of this domain may not be the sole determinant is suggested by a separate study [37°°], which identified a cysteine and a histidine residue in the cytoplasmic half of the transmembrane domain that are important for the Golgi retention of GT. There is no apparent sequence similarity between the transmembrane domains of these glycosyltransferases.
Golgi retention of a coronavims E1 protein has also been found to be determined by the first of its three membrane-spanning domains [38.°]. Replacing the transmembrane domains of two proteins normally destined for the plasma membrane with this domain leads to their retention in the Golgi. Polar uncharged residues in this transmembrane domain, which would line up on one face of an a-helx, are well conserved among coronaviruses. Mutation of these residues to isoleucine, or insertion of two isoleucines to disrupt the helical periodicity, results in the transport of some of the protein to the plasma membrane. These data should be compared with those obtained with the E1 protein of the mouse hepatitis virus A59 [39°°]. In this case, the data obtained suggest that the Golgi retention signal is a more general property of the molecule. The reasons for this difference are unclear.
Another intriguing case of potential interactions between intramembraneous domains is that involving the 44-amino-acid E5 oncogene product of fibropapillomaviruses. The amino-terminal two thirds of E5 has a sequence suggesting a transmembrane a-helix, whereas the 14-amino-acid hydrophilic carboxy-terminal tail extends into the lumen of the Golgi apparatus (for a review, see [40]). A conserved glutamine residue within the hydrophobic domain is important for association with the hydrophobic 16kD subunit of the vacuolar H+-ATPase [41o]. However, there appear to be no spe-cific sequence requirements in the remainder of the hydrophobic region for this association to occur. The glutamine residue is also important for the transforming ability of ES. For this activity, however, there are specific sequence requirements in the hydrophobic domain of E5 [42.-]. The transmembrane domain of E5 may also serve as its Golgi retention signal.
The formation of bundles of transmembrane ~-helices appears to occur in some other cases. One such example is the 52-amino-acid protein phospholamban, which is a regulator of the Ca 2 + -ATPase of cardiac muscle sarcoplasmic reticulum. Phospholarnban forms pentamers that are stable in sodium dodecyl sulichte (SDS), although the relevance of this to "its function is not yet clear. Residues within the predicted transmembrane ct-helix have been shown to be involved in pentamer formation [43]. In the case of ion-conducting channel proteins, the pore itself may consist of a bundle of amphipathic or-helices. Specific peptide sequences, which it has been predicted would form the channel-lining helices, have been synthesized, and shown to reproduce a number of properties of the channels from which their sequences were derived (for a review, see [44] ).
Perhaps the best characterized example of interactions between transmembrane or-helices is the human erythrocyte sialoglycoprotein glycophorin A (GpA), which forms a dimer that is stable in SDS. Bormann et al. [45] showed that the GpA dimer was disrupted upon addition of a synthetic peptide corresponding in sequence to its transmembrane domain. The addition of a number of heterologous transmembrane peptides did not disrupt the dimer. Further studies using a chimeric protein have shown that the GpA transmembrane domain alone contains all of the information required for this specific association [46..]. In addition, mutational analysis of the chimera shows that even very subtle alterations in the nature of certain side chains can significantly disrupt the interaction. For example, mutation of a valine to a leucine in the transmembrane domain disrupts the dimerization, and therefore this residue is proposed to lie at the dimer interface. If the or-helix geometry is canonical as CD data would seem to suggest, this valine would lie on the same face of the helix as several of the glycine residues in the transmembrane domain, There are no highly polar residues in the transmembrane domain of GpA, which thus serves as a contrast to the cases mentioned above.

Intramembraneous helix-helix interactions in transmembrane signalling by receptors
It is now widely accepted that the primary event in transmembrane signalling by receptors such as the epidermal growth factor receptor (EGF-R), is ligand-stimulated receptor dimerization (for a review, see /47,48..]. Since it was found that the mutation of a valine to glutamic acid in the transmembrane domain of the neu oncogene product causes this EGF-R-Iike receptor to become constitutively active as a tyrosine kinase [49] and increases the proportion of the receptor existing as a dimer [50], there has been much speculation concerning the possible role of interactions between transmembrane cz-helices in this signalling mechanism [51"']. A number of experiments have been reported in which transmembrane domains of related proteins have been swapped, resulting in the production of inactive receptors. For example, Yan et al. [52"] made chimeric receptors in which the ectodomain was derived from EGF-R, and the intracellular domain was from the low-affinity nerve growth factor receptor (NGF-R) represented by the p75 NGFR protein. A morphological response to epidermal growth factor (EGF) stimulation was seen for PC12 cells expressing a chimera in which the transmembrane domain was derived from the p75 NGF-R, but not if it was derived from the EGF-R. The low-affinity p75 NGF-R may interact with the trk oncogene product (pl40pr°t°trk), another low-affinity NGF-R, to form high-afflnity binding sites for NGF [53"]. The experiments of Yan et al [52"], and the fact that the sequence of the transmembrane domain of p75 NGF-R is highly conserved between species [54], suggest that this predicted transmembrane cz-helix may be involved in interactions with other proteins, such as pl40P r°t°trk.
Based upon consideration of the amino acid sequence surrounding the activating Val--+Glu mutation in the transmembrane domain of the neu oncogene product, and comparison with a similar region of other related receptors, a model has been proposed in which a fiveamino-acid motif is responsible for the specific dimerization of transmembrane a-helices in a number of receptors of this type [55]. A recent mutational analysis of the neu oncogene product has confirmed that a subdomain within its transmembrane domain, consistent with the nature of this motif, is required for activation of transformation by the Val--+Glu mutation mentioned above [56"]. In contrast with the conclusions drawn from this study, analysis of the effects of deletions and mutations in the transmembrane domain of the insulin receptor [57"] and EGF-R [58",59] seems to indicate a passive role for these domains in signal transduction by these receptors. Furthermore, the extracellular and transmembrane domains of v-erbB, the truncated and constitutively active form of the EGF-R, can be replaced by a myristyl anchor without affecting transformation potency or specificity [60,]. This argues against the dimerization of the transmembrane domain of EGF-R being important in activating the tyrosine kinase domain of this receptor.
Whatever their exact role, transmembrane domains must be important in signal transduction, as they comprise the only connection between the ligand-binding domain of the receptor and the effector region in the cytosol. Recent studies of the chemotactic aspartate receptor (Tar) of E. colimay shed some light upon this issue. The structure of the ectodomain of this dimeric receptor, both with and without ligand, was reported last year [61".], and showed a small ligand-induced rotation of the subunits about an axis parallel to the membrane. It is proposed that this rotation is translated to a relatively large alteration in the relative disposition of the endodomains of the dimer, resuiting in their activation. An extensive analysis of disulfide crosslinking of cysteine-substituted proteins within the membrane region of this receptor by Pakula and Simon [62.-] has provided data for the construction of a model in which the four transmembrane domains (two from each transmembrane subunit; TM1 and TM2) form a distorted four-helix bundle, the two TM1 helices interacting the most extensively. Residues capable of participating in crosslink formation were found to be restricted to one face of each helix, implying that the TM1-TMI' interaction is axially symmetric. The face of each helix thus shown to be involved in helix-helix interactions was identified as the most hydrophilic and most conserved face [63.o], using the analytical method of Rees et al. [7]. For example, there are glutamine and serine residues in the TM1-TMI' interface, which may participate in interhelical hydrogen bonding. It will be very interesting to see how the pattern of disulfide crosslinking changes in the aspartate-stimulated receptor. Preliminary data suggest that the two TM1 helices are closer together in the activated receptor [63o-].

Conclusions and perspectives
Many generalizations have been put forward for membrane helix properties during the past decade. Some of these apply relatively broadly, others more specifically; few describe the full range of properties without exception. A number of notions that are useful from a structural perspective now exist. The side chains of a transmembrane a-hdix are largely hydrophobic. A limited number of potentially charged groups may be included, although it is not clear that they would be ionized within the bilayer. Proline residues occur more commonly in the transmembrane a-helices of polytopic membrane proteins than in cz-helices of soluble proteins, and may cause a kink in many cases. Where transbilayer helices are significantly amphipathic, the more polar surface is likely to be involved in interactions with other helices and prosthetic groups rather than with the lipids. Association of cz-helices within the bilayer may be driven by strong polar interactions and/or detailed van der Waals fits. These interactions can be highly specific, and can have sufficient energy to drive association between helices without covalent linkages outside the membrane.
On the functional side, a rapidly expanding list is becoming established, including roles in defining channels arrd transmembrane transport pathways, signals for oligomerization and for degradation if oligomerization fails, positioning of prosthetic groups for electron-transfer reactions, signals for selective localization in specific membrane compartments, and as mediators of transmembrane signalling. It is clear that transmembrane helices are more than mere hydrophobic anchors. It seems a reasonable hope that the future will allow an understanding of the function of these important structural elements that will permit a chemical understanding of the myriad functions that they perform. Plant Light-harvesting Complex Determined by Electron Crystallography. Nature 1991, 350:130-134. This paper describes the structure of light-harvesting complex II at 6,~ resolution, the third example of a helical membrane protein structure determination with resolved secondary structure. The trimeric protein has three transmembrane helices and binds several chlorophyll molecules. Two of the three helices are rather longer than those seen in bacteriorhodopsin or RC, and associate as in a right-handed supercoil, with an included angle of -56 °. The third helix, helix C, is of the expected length for a transmembrane cx-helix, and interacts largely with chlorophyll molecules. Subunit contacts in the trimer appear to be defined both by helix-helix contacts and helix-chromophore contacts. author points out that where proline residues occur in the middle of transmembrane co-helices, the helices are kinked. Furthermore, the convex face of the kinked helix tends to be buried against other helices in the protein. This conclusion is based upon analysis of the structures of bacteriorhodopsin, which contains three prolines, and the RC of Rhc~opseudomonas viridis, which contains one central proline and several others located towards the ends of the helices. In addition, proline-containing transmembr'ane a-helices predicted by sequence analysis were studied. The presumed convex side of such predicted helices tends to be the most polar, and hence the most likely to interact with other parts of a membrane protein.

11.
WOLFSON DN, MORTISHIRE-SMITH RJ, WILLIAMS DH: Conserved • Positioning of Proline Residues in Membrane-spanning Helices of Ion-channel Proteins. Biochem Biophys Res Commun 1991, 175:733-737. An analysis of the structural role of proline residues in membrane-spanning a-helices, similar to that described in [10•], which is restricted to helices of ion-channel proteins. The analysis suggests that proline residues are often found on the hydrophilic faces of pore-forming helice,. A single basic or acidic residue introduced into the transmembrane domain of a protein normally destined for the cell surface (Tac) targets this protein for retention and degradation in the ER. The introduction of arginine or aspartic acid in regions dose to the center of the tmnsmembrane sequence is found to be most effective in this regard.

24.
COSSON P, LANKFORD SP, BONIFACINO JS, KIAUSNER RD: Memoo brahe Protein Association by Potential Intramembrane Charge Pairs. Nature 1991, 351:414-416. Two basic residues in the transmembrane domain of TCR~ are shown to be of prime importance for its association with CD38, which has an aspartic acid residue in its transmembrane domain. The TCR donotypic chain, which contains the same basic residues as TCR0t, is also shown to associate with CD3~. Furthermore, a mutant of Tac, in which an arginine had been introduced into its transmembrane domain, is shown to interact with CD3& This mutant is degraded in the ER [23"']. Mutation of the aspartic acid residue in CD3fi abrogates all of these associations. These data suggest that interl~elical salt bridges and/or strong hydrogen bonds are involved in the assembly of the TCR complex.  1991, 88:3837-3841. This paper presents a demonstration that the ~-subunit of TCR/CD3 associates through interactions mediated by its transmembrane domain with the cx-subunit of the FcyRIII receptor (CD16) of human natural killer cells. CD16 is thus spared from degradation in the ER. An aspattic acid residue in each of the transmembrane domains is important for this interaction, as is a leucine residue in the transmembrane domain of 4. Mutation of this leucine to isoleucine reduces the extent of interaction by 65%. Immunofluorescence microscopy is used to study the location in COS cells of variants of a chimera between GT and the human chorionic gonadotrophin 0c-subunit. A variety of replacement mutations have no effect upon subcellular localization; however, the cytoplasmic half of the transmembrane domain of GT does seem to be important in this regard. In particular, a cysteine and a histidine residue in this region are found to be important for Golgi retention. This result should be compared with that obtained in [36"*[, where the lumenal half of the transmembrane domain is identified as being the region most important for Golgi retention.

38.
SWIFT AM, MACHAMER CE: A Golgi Retention Signal in a ** Membrane-spanning Domain of Coronavirus E1 Protein. J Cell Biol 1991, 115:19-30. The first of the three transmembrane domains of the E1 protein of the avian coronavims infectious bronchitis vires is substituted for the transmembrane domains of two other transmembrane proteins which are normally transported to the cell surface. Both proteins are consequently retained in the Golgi apparatus. Point mutations within this transmembrane domain which alter the periodicity of conserved polar uncharged residues, which would usually line up on the same face of an assumed canonical co-helix, abrogate Golgi retention. Disruption of the relative spacing of these residues by insertion of two isoleucine residues has the same effect. The results from this study should be compared with those in [39"*].

39.
ARMSTRONG J, PATEL S: The Golgi Sorting Domain of Coronavirus E1 Protein. J Cell Sci 1991, 98:567-575. nthe E1 protein of the coronavirus mouse hepatitis virus A59, studies of the signals for Golgi retention give rather different results from those described in [38**] for the avian infectious bronchitis virus. In this case, the retention signal appears to comprise several portions of the El protein, rather than a single peptide sequence. The discussion in this paper also highlights the problems inherent in interpreting studies of localization signals through the construction of chimeric proteins.

40.
PETrl L, NILSON L, KULKE R, LEPTAK C, RIESE DJ, ZIBELLO "T,  Annu Rev Biophys Biomol Struct 1992, 21:233-242. This review discusses the two-stage model for integral membrane protein folding as it applies to those proteins with a single transbilayer ~-helix. It further considers models for transmembrane signalling by such proteins. A model involving receptor oligomerization is argued to be most reasonable, and evidence supporting such a model, and the role of transmembrane domains in ollgomerization, is reviewed.
tors Define Domains Responsible for Neuronal Differentiation. Science 1991, 252:561-563. Chimeric receptor molecules are constructed in which the endodomain is derived from the p75 NGF-R and the ectodomain from EGF-R. Responses similar to those usually seen with NGF are observed upon EGF stimulation of cells expressing these chimerae only when the transmembrane domain of the receptor is derived from p75 NGF-R. Thus, it is considered that this domain may be involved in interaction with other NGF*responsive signal-transducing molecules, such as the irk oncogene product.

53.
HEMPSTEAD BL, MARTIN-ZANCA D, KAPLAN DR, PARADA IF, CHAO * MV: High-afflnity NGF Binding Requires Coexpression of the trk Proto-oncogene and the Low-affinity NGF Receptor. Nature 1991, 350:678-683. This paper describes experiments demonstrating that p75 NGF-R and pl40P r°totrk, each of which form low affinity binding sites for NGF when present alone, form a high-affinity NGF binding site when present together. Fusion of membrane preparations from cells expressing just pl40P r°t°trk with similar preparations from cells expressing just p75 NGF-R led to the appearance of both high-and low affinity NGF binding sites, whereas the individual preparations had just low-affinity binding sites. Transient transfection of COS cells with plasmids expressing both proteins also indicated that co-expression of the two proteins is required for high-affinity NGF binding. Thus, it is proposed that p75 NGF R and p140P r°t°trk interact to form the high-affinity NGF-R. in the Transmembrane Domain is Necessary for p185 neu Activation. EMBO J 1992, I 1:923-932. A number of mutations are made in the transmembrane domain of the neu oncogene. In the context of the activating Val-+Glu mutation at position 664, it is found that the transforming ability of the oncogene product is in general resilient to alterations. However, alteration of residues at positions 661~o65 inhibits transforming ability. Furthermore, replacement of the carboxy-terminal two thirds of the neu transmembrane domain with that of CD4 abrogates transformation. Although it is difficult to draw conclusions from such a limited set of mutations, this work clearly shows that there are determinants within the transmembrane domain that are important for the transforming ability of this protein. This result contrasts with those obtained for EGF-R [58%59] and the insulin receptor [57"]. It should be noted, however, that, in the absence of an available ligand for the neu oncogene, it was not possible to determine the ability of the mutants in this study to transduce signals across the membrane.
a Passive Role for the Insulin Receptor Transmembrane Domain in Insulin-dependent Signal Transduction. J Biol Cbem 1991, 266:98290834. A series of mutations are made in the transmembrane domain of the insulin receptor, and their effect upon insulin-stimulated receptor autophosphorylation are analyzed. Deletion of up to five amino acid residues from within the transmembrane domain is shown to have no effect upon insulin-stimulated signalling. Furthermore, the introduction of a mutation analogous to that which activates the neu oncogene has no detectable effect. Thus, these data, although rather limited, lead to the opposite conclusion from that drawn in [56*]. Mutations are made in the transmembrane domain of EGF-R, and their effects upon EGF binding, EGF-stimulated tyrosine kinase activity, and EGF-dependent dimerization are analyzed. Shortening of the transmembrane domain by six residues from its carboxyl terminus, by 10 from its amino terminus, or the insertion of three proline residues separated from one another by four residues, has no effect upon these activities of the EGF-R. Furthermore, mutations analogous to those found to activate the neu oncogene are found to have no such effect upon the EGF-R. This work complements that reported in [59], drawing the sim ilar conclusion that the transmembrane domain of the EGF-R plays a passive role in signal transduction across the bilayer. Although limited, these data suggest that the situation with EGF-R may be different from that seen for the neu oncogene product [56"]. . It is shown that the transmembrane and extracellular domains of v-erbB are not necessary for its transforming ability. Rather, what seems to be necessary is that the cytosolic domain is anchored in the membrane. In order to achieve this, the amino-terminal 14 amino acids of v-src, which contain a myristylation signal, are used to replace these domains. The resultant myristyl anchored v erbB cytosolic domain retains the transforming properties of v-erbB. . The X-ray crystal structure of a disulfide-crosslinked dimer of the ectodomain of Tar is reported with and without bound ligand (at 2.0 and 2.4A. resolution, respectively). The protein is essentially a dimer of four-helix bundles, which suggests that a four-helix bundle transverses the lipid bilayer. One aspartate molecule binds to the dimer, causing a conformation change which can be described as a 4 ° rotation between the subunits about a pivot axis perpendicular to both the dimer interface and the dimer twofold axis. Such a change could be transmitted to the cytosolic domain via movement of the transmembrane ~x-helices with respect to one another, leading to activation of that domain. Through an extensive analysis of the positions within the transmembrahe region between which disulfide crosslinks will form, data are generated for modelling of the relative disposition of the four transmembrane helices of the receptor dimer. The positions which crosslink most readily are found to lie on one face of each helix of each subunit (TM1 and TM2). The two TM1 helices of the dimer interact extensively with one another in a distorted four-helix bundle structure suggested by the modelling. The TM2 helices interact less extensively. The most highly conserved transmembrane residues are found to occur at or near the helix-helix interfaces inferred from the crosslinking study.

63.
LYNCH BA, KOSHLAND DE: Disulfide Cross-linking Studies of .• the Transmembrane Regions of the Aspartate Sensory Receptor of Escherichia coli. Proc Natl Acad Sci USA 1991, 88:10402-10406. A rather more limited set of cysteine substitutions is studied in this paper, yielding results which are consistent with those of [61"]. Of particular interest is the finding that the rate of crosslink formation for two TM1 TMI' crosslinks is increased in the presence of aspartate. This indicates that these helices are drawn closer or have a different relative orientation in the activated receptor. An analysis of TM1 and TM2 according to the method of Rees et aL [7] is also presented, from which it appears that the most hydrophilic and most conserved face of each helix is that most intimately involved in helix-helix interactions.
MA Lemmon and DM Engelman, Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 6666, 260 Whitney Avenue, New Haven, Connecticut 06511, USA.