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Mapping Structural Determinants within Third Intracellular Loop That Direct Signaling Specificity of Type 1 Corticotropin-releasing Hormone Receptor*

Background: The CRH-R1 is key for mammalian adaptation to stress; however, the structural determinants of signal transduction are unknown. Results: Amino acids identified within CRH-R1 IC3 appear crucial for G protein-dependent cAMP and ERK1/2 signaling. Conclusion: These microdomains provide a tight control of CRH-R1 differential coupling to distinct G proteins and downstream effectors. Significance: This might prevent hyperstimulation of stress-induced responses.


data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N-and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.
The type 1 corticotropin-releasing hormone receptor (CRH-R1) 3 belongs to the class B1 subfamily of heptahelical receptors ("brain-gut" neuropeptide receptors) that transduce signals primarily, but not exclusively, through activation of G proteins (i.e. G protein-coupled receptors, GPCRs). The CRH-R1 is widely expressed in the brain and peripheral tissues and orchestrates mammalian homeostatic adaptation mechanisms to stressful stimuli (1). The CRH-R1 elicits a wide range of biological effects, sometimes in a tissue-specific manner, via activation of a variety of signaling pathways using diverse G proteins (2). A number of downstream signal effectors for CRH-R1 have been described; however, the cAMP and the extracellular signal-regulated kinase (ERK)1/2 pathways represent the principal signals that mediate CRH-R1 action after interaction with agonists such as CRH and urocortin 1 (Ucn1) in target cells (3,4).
A variety of molecular mechanisms modify CRH-R1 signaling. Previous studies have shown that protein kinases such as PKA mediate cross-talk between signaling pathways via phosphorylation of specific amino acid residues in the third intracellular loop (IC3) of CRH-R1, thereby regulating efficiency of receptor coupling to G q protein and downstream ERK1/2 signaling (5). G protein-coupled receptor kinases also influence CRH-R1 action through amino acid phosphorylation that leads to desensitization and subsequent endocytosis of receptor endocytosis (6), a mechanism that can either reduce specific signaling cascades such as the adenylyl cyclase (AC)/cAMP pathway or activate ERK1/2 and p38MAPK cascades (7). Specific Ser/Thr phosphoacceptor residues have been identified in both the IC3 and the distal segment of the C terminus of CRH-R1 (6,8). Phosphorylation of the latter domain, especially at position Thr 399 , appears to be important for ␤-arrestin2 recruitment and desensitization of cAMP signaling (6). Interestingly, mechanisms leading to desensitization do not affect coupling of CRH-R1 to all classes of G proteins (9). Receptor conformations that activate G s and G q / 11 proteins are sensitive to desensitization, whereas G i proteins are activated by different receptor states that do not promote signaling desensitization.
The domains that determine coupling of CRH-R1 coupling to distinct G proteins and downstream signaling pathways are not well understood. Most GPCR dynamic functions such as receptor activation, internalization, desensitization, and resensitization involve key structural determinants present in the cytoplasmic loops and carboxyl tail (10 -13). However, the extensive structural diversity among GPCRs suggests that efficient G protein coupling can be achieved through various unrelated structural motifs. In fact, conformation and charge rather than primary sequence appear to be the major determinants for G protein activation. Past studies have identified the IC3 as a key element that regulates the ability of GPCRs to activate G proteins and trigger intracellular responses (14,15). Within IC3, the juxtamembrane regions of IC3 that form cationic ␣-helical structures (16) appear to be important for the activation of distinct unrelated G protein subtypes. Moreover, the IC3 appears to be important for G protein-independent signaling of GPCRs; for example the IC3 N-terminal part of CXCR4 is required for JAK2 activation leading to phosphorylation of only one cytoplasmic Tyr residue, present at the C terminus of IC2, which triggers STAT3 activation (17).
In the present studies we sought to identify the amino acid residues within IC3 that are critical for CRH-R1 signaling. We therefore introduced tandem and single alanine mutations by PCR-mediated mutagenesis, replacing amino acid cassettes at the juxtamembrane regions of IC3 or specific residues including basic amino acids (Arg 292 , Lys 297 , Arg 299 , Arg 310 , Lys 311 , and Lys 314 ), and assessed the ligand binding and signaling characteristics of modified receptors. Our results demonstrate that both N-and C-terminal regions of the IC3 of CRH-R1 as well as basic amino acids possess specific structural elements with distinct contributions in G protein coupling and downstream cAMP production and ERK1/2 activation.
Transfection of CRH-R1 to HEK293 Cells-Lipofectamine 2000 reagent was used for transfection of HEK293. Transient transfections were performed as described previously (18) when cells seeded in 25-cm 2 vented flasks reached 50 -70% confluence. Approximately 24 h after transfection, cells were transferred to poly-D-lysine (Sigma)-coated glass coverslips (for confocal microscopy studies) or to 12-well plates for signaling studies. The experiments were performed 48 h after transfection. Receptor expression was verified by confocal microscopy analysis.
CRH-R1 Mutagenesis-A series of CRH-R1␣ mutants were generated by overlap extension PCR using high fidelity Pfu polymerase and mutagenic primers (supplemental Table 1) as described previously (20). The mutagenic CRH-R1 cDNA was cut sequentially with EcoRI and HindIII and then ligated back into the original pcDNA3.1(Ϫ). All mutant cDNAs were confirmed by sequence analysis of both strands (Molecular Biology Service, Department of Biological Sciences, University of Warwick).
Signaling Assays-ERK1/2 activation in response to 100 nM Ucn1 was determined in transiently transfected HEK293, seeded in 12-well plates to reach 60 -70% confluence, as described previously using the Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK), (19). Cyclic AMP production was measured as described previously (19) by a commercially available ELISA Direct Cyclic AMP Enzyme Immunoassay kit (Assay Designs Inc., Ann Arbor, MI). In some experiments Ucn1-induced cAMP production was determined following pretreatment of cells with either pertussis toxin (PTX) for 12 h, (final concentration, 100 ng/ml) or 20 mM mastoparan for 1 h, or the suramin analog NF023 (250 nM for 2 h).
In some experiments, AC activity and inositol trisphosphate (IP 3 ) production were assessed using plasma membranes of HEK293 cells expressing wild-type or mutant receptors as described previously (22). Briefly, cell membrane preparations (25 g of protein) were incubated in triplicate with Ucn1 (100 nM) at 22°C in the presence of 100 l of 50 mM Tris-HCl buffer containing 10 mM MgCl 2 , 1 mM EGTA, 1 g/liter BSA, 1 mM ATP, ATP regeneration system (7.4 mg/ml creatine phosphate, 1 mg/ml creatine phosphokinase), 100 M 3-isobutyl-1-methylxanthine, pH 7.4. The reaction was terminated after 15 min by the addition of 1 ml of 0.1 M imidazole buffer, pH 7, followed by heating at 95°C for 5 min. Cyclic AMP was measured by ELISA. For the IP 3 assay, cell membrane suspensions (40 g) were incubated with increasing concentrations of Ucn1 followed by the addition of 200 l of IP 3 generation buffer containing 25 mM Tris actetate buffer, pH 7.2, 5 mM Mg acetate, 1 mM DTT, 0.5 mM ATP, 0.1 mM CaCl 2 , 0.1 mg/ml BSA, and 10 M GTP. Membranes were incubated for 3 min at 37°C, and the reaction was terminated by the addition of 1 M ice-cold trichloroacetic acid, followed by extraction of inositol phosphates and neutralization. IP 3 levels were estimated by a radioreceptor assay (PerkinElmer Life Sciences) based on the displacement of [ 3 H]IP 3 from calf cerebellum-derived membranes containing the IP 3 -binding protein.
Plasma Membrane Fractionation and Co-immunoprecipitation Studies-For plasma membrane fractionation, cells were grown in 10-cm Petri dishes, and when 80% confluence was reached, cells were deprived of FCS for 1 h before treatment with 100 nM Ucn1 for 5 min. Membrane protein extracts were prepared using a ProteoExtract Native Membrane Protein Extraction kit (Calbiochem/Merck Biosciences). Protein concentration was determined using a BCA Protein Assay kit (Pierce). Protein fractions (5 g) were analyzed by Western blot analysis using CRH-R1/2 antibodies (Santa Cruz Biotechnology) (dilution 1:100). Antibody binding was detected using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Dako) and ECL reagents from Amersham Biosciences. The relative density of the bands was measured by optical density scanning using the software Scion Image-Beta 3b for Windows (Scion Corp., Frederick, MD).
For co-immunoprecipitation studies, plasma membrane fractions were incubated overnight at 4°C with antibodies specific for CRH-R1/2 (Santa Cruz Biotechnology) to a final concentration of 1:100 and protein A/G-agarose. The immunoprecipitates were washed four times in PBS, and proteins were eluted from the agarose beads by heating at 95°C in Laemmli sample buffer. The resuspended proteins were analyzed by electrophoresis through 10% SDS-polyacrylamide gels and then electrophoretically transferred to PVDF membranes. Following blocking with 5% nonfat dry milk at room temperature for 30 min, membranes were subjected to immunoblot analysis by incubation at 4°C for 2 h with G␣ i1/2 protein-specific antisera (dilution 1:200). After incubation with the secondary HRPconjugated anti-rabbit IgG, proteins were visualized with ECL.
Data Analysis and Statistics-The relative involvement of G␣ s and G␣ i proteins on AC activation in HEK293 cells, transfected with wild-type or mutant CRH-R1␣, was quantified as follows: cAMP responses (measured as Ucn1-induced cAMP -fold increase above basal) in control cells (R T ) were considered as responses involving both G␣ s ϩ G␣ i ; cAMP responses in cells pretreated with PTX that inactivated G␣ I proteins were considered as G␣ s -exclusive responses (R s ). The G␣ i protein contribution (R i ) was calculated by using the equation: R i ϭ R s Ϫ R T. The results obtained are presented as the mean Ϯ S.E. of each measurement. Data were tested for homogeneity, and comparison between group means was performed by one-or two-way ANOVA. Probability values of p Ͻ 0.05 were considered significant.

Binding Properties of Mutant CRH-R1␣
Receptors-To identify residue(s) in the CRH-R1␣ N-and C-terminal regions of IC3 involved in receptor signaling, we used a tandem alanine scanning mutagenesis strategy, in which four consecutive amino acid residues were replaced by alanines (Fig. 1). A series of additional mutants was also generated containing single Ala substitutions of residues in the proximal (Arg 292 -Met 295 ) and distal (Lys 311 -Lys 314 ) terminal segments, as well as the charged amino acids Lys 297 , Arg 299 , Arg 310 . Mutant and wild-type receptors were transiently expressed in HEK293 cells, and immunoblotting experiments using a specific CRH-R1/2 Ab demonstrated that levels of expression at the cell membrane were similar (supplemental Fig. 1). Most mutants retained high binding affinity for the radiolabeled agonist 125 I-Tyr 0 -sauvagine (supplemental Table 2), comparable with the wild-type CRH-R1␣ (data not shown), indicating retention of the normal conformation of the wild-type receptor. However, mutant CRH-R1␣(N-4A/C-4A), where both N-and C-juxtamembrane regions of IC3 were substituted to Ala, showed a 5-8-fold Mutant receptors were generated with single or tandem amino acid Ala substitutions within the predicted IC3 amino acid sequence. The mutants were generated by overlap extension-PCR as described under "Experimental Procedures." An alanine residue was preferred due to lack of amino acid side chain beyond the carbon and thus able to determine the importance of specific amino acid side chains for receptor-G protein interactions. Furthermore, Ala substitution avoids potential problems associated with steric hindrance or unwanted ionic interactions, when amino acid residues with bulky side chains or charged groups are introduced.
increase in IC 50 compared with the wild-type receptor (8.79 Ϯ 2.93 versus 1.26 Ϯ 0.76 nM, respectively), indicating reduced ligand binding affinity. Cell membranes expressing wild-type or mutant CRH-R1␣ receptors exhibited comparable levels of total amount of binding sites (B max ) available for agonist binding (average 865 Ϯ 60 ranging from 714 Ϯ 36 to 980 Ϯ 135 fmol/mg of protein, respectively, as determined by Scatchard analysis of 125 I-Tyr 0 -sauvagine binding to plasma membranes) (supplemental Table 2). By contrast, the B max of overexpressed CRH-R1␣(N-4A/C-4A) mutant was also found to be decreased by 15-20% (p Ͻ 0.05).
Structural Motifs within IC3 of CRH-R1␣ IC3 Involved in Activation of AC-Our previous studies (26) demonstrated that although the CRH-R1␣ can activate multiple classes of G proteins, it is most efficiently coupled to G␣ s proteins leading to downstream activation of AC and elevation of intracellular cAMP levels (24). The role of the juxtamembrane regions of the IC3 predicted amino acid sequence in Ucn1-induced cAMP production was further investigated in HEK293 cells transiently expressing wild-type or mutant CRH-R1␣. Introduction of tandem Ala mutations in either the Arg 292 -Met 295 or Lys 311 -Lys 314 terminal regions of the IC3 (mutants CRH-R1␣(N-4A) and CRH-R1␣(C-4A), respectively) significantly increased maximal cAMP production stimulated by Ucn1, by 78 and 87% respectively, compared with the wild-type receptor (Fig. 2a). These mutations did not substantially alter the EC 50 values of mutant receptors (supplemental Table 3). However, mutant CRH-R1␣(N-4A/C-4A) demonstrated a 90% decrease in cAMP response compared with the wild-type receptor, suggesting that disruption of IC3 secondary structure severely impaired receptor ability to couple to G proteins crucial for AC activation. Interestingly, the N-and C-tandem Ala mutations altered the shape of agonist dose-response curves; although the wildtype receptor exhibited a biphasic pattern of responses where maximum cAMP production at 100 nM Ucn1 was followed by a slightly reduced response at higher agonist concentration (1 mM), mutant receptor responses were characterized by a plateau at the maximum response with no apparent reduction observed at higher Ucn1 concentrations. These changes might reflect alterations (reduction) in inhibitory G protein contribution to AC activation in the mutants compared with the wildtype receptor at high Ucn1 concentrations.
To identify specific amino acid residues important for G protein coupling and AC activation, we examined agonist-induced cAMP responses in a series of mutant CRH-R1␣ receptors that contained single Ala substitutions throughout the N-and C-terminal segments of IC3 as well as other charged amino acids. We found that single Ala substitution of any of the five charged amino acids (Arg 292 , Lys 297 , Arg 310 , Lys 311 , or Lys 314 ) significantly increased Ucn1-induced maximal cAMP production, without affecting EC 50 values of cAMP responses (EC 50 (nM) ϭ 4 Ϯ 0.8) (Fig. 2b). Receptor activation of cAMP was increased by 70% over wild-type when Arg 292 or Lys 311 was replaced by alanine whereas Lys 297 , Arg 310 , or Lys 314 substitution led only to a 50% increase in cAMP production. A significant increase in cAMP response, albeit to a lesser degree, was also observed when either Lys 294 or Met 295 was replaced by alanine (25 and 30% increase of wild-type receptor cAMP response, respectively). In contrast, Ala substitution of Arg 299 reduced by 20 -25% maximum cAMP production, again without affecting EC 50 value of cAMP response.
Previous studies (4) suggested that the CRH-R1 can adopt distinct, agonist-specific, active conformations that result in important differences in G protein interaction and downstream signal effects. Hence, we used CRH and Ucn1 to activate CRH-R1␣ mutants to investigate whether Ala substitutions resulted in agonist-specific differences on receptor ability to stimulate AC activity. For all mutants tested (R292A, L294A, M295A, R299A, K311A, and K314A), no significant differences were found between CRH and Ucn1 maximal (Fig. 3) or EC 50 (supplemental Table 3) values of cAMP responses, suggesting a similar role for these amino acid residues in the receptor active conformation, induced by CRH and Ucn1, required to activate the pathway leading to increased intracellular cAMP levels. Similar agonist responses were observed when mutants I293A, K297A, and R310A were transiently expressed in HEK293 cells (data not shown).
In addition to high affinity coupling to G␣ s , CRH-R1␣ receptors can also couple to G␣ i proteins especially at nanomolar agonist concentrations (9,26). Hence, it is likely that the net activation of AC reflects the opposing actions of these G pro-  MARCH 2a) demonstrated that at agonist concentrations greater than 1 nM, PTX pretreatment enhanced Ucn1-induced AC activation, without any change in the bell-shaped dose-response curve; this suggests a role for G i/o -mediated inhibition of AC activation in addition to G s -dependent stimulation, especially at high agonist concentrations that correspond to the G i -coupled phase, as described previously (9). By contrast, at subnanomolar concentrations Ucn1 effects were exclusively mediated via G s -induced AC activation, a result consistent with the lower coupling affinity of the receptor to G i proteins (25). The involvement of G i/o proteins in receptor-mediated cAMP responses was also investigated in experiments employing mastoparan, the wasp venom-derived peptide, which can selectively activate G i/o proteins (27). Mastoparan pretreatment attenuated Ucn1-induced cAMP responses in control cells (supplemental Fig. 2a), consistent with receptor-independent activation of G i/o proteins and inhibitory actions on Ucn1-induced AC activation. However, mastoparan had no effect in cells expressing a G␣ i dominant negative (DN) (Q205L/D273N) (supplemental Fig. 2b), thus providing further evidence that G i/o proteins are involved in mastoparan regulation of AC activity. Ucn1-induced cAMP responses were also sensitive to NF023, a selective G i/o protein inhibitor, and cells pretreated with 250 nM NF023 exhibited increased agonist-stimulated cAMP responses (supplemental Fig. 2c). Interestingly, the bellshaped response remained detectable even after treatment with either PTX or mastoparan, suggesting that the inhibitory phase was partially independent of receptor coupling to G i .

IC3 Structural Motifs in CRH-R1 Signaling
These results led us to examine the possibility that the enhanced cAMP responses observed in mutants R292A, K297A, R310A, K311A, and K314A or the reduced cAMP response of R299A was due to altered coupling of receptors to G␣ s and/or G␣ i proteins. Cells expressing wild-type or mutant receptors were pretreated with or without PTX (to inactivate G i/o proteins) prior to agonist stimulation and AC activation. The effectiveness of PTX in inactivating G i/o proteins has been previously confirmed by demonstrating inhibition of agonist-induced labeling of G␣ i proteins with the nonhydrolyzable GTP analog [␣-32 P]GTP-AA (25). Results (Fig. 4aI) showed that wild-type CRH-R1␣ activation by 100 nM Ucn1, to induce maximal cAMP production, involved G␣ s proteins as well as PTXsensitive G proteins; the latter exerted an inhibitory effect (by 18% Ri component) on AC activity, as demonstrated by an increased agonist-induced (but not basal) cAMP response in PTX-treated cells that unmasked the full activity of G␣ s proteins. We used the same experimental paradigm in cells expressing receptor mutants, which exhibited altered cAMP responses (Fig. 2). In K314A-expressing cells (Fig. 4a), we found that PTX pretreatment enhanced Ucn1-induced cAMP production by 20% above control (untreated) cells, indicating similar to the wild-type, R i responses. This suggests that the alanine substitution at position Lys 314 had negligible effects on receptor G␣ i protein coupling and that the increased R s response resulted from a more efficient coupling to G␣ s proteins. By contrast, in CRH-R1␣(R292A), CRH-R1␣(K297A), CRH-R1␣(R310A), and CRH-R1␣(K311A), uncoupling of G␣ i protein activation by PTX pretreatment did not significantly modify cAMP production in response to 100 nM Ucn1 (Fig. 4b), suggesting that these mutant receptors exhibited minimal G␣ i protein coupling. Similarly, CRH-R1␣(R299A) response was not affected by PTX pretreatment, suggesting negligible G␣ i protein coupling and contribution to the overall cAMP response (Fig. 4aII). Thus, it appears that this mutation can attenuate G␣ i protein coupling as well as reduce receptor R s response, suggesting that the Arg 299 residue is required for optimal interaction with G␣ s proteins.
To confirm the increased G␣ s protein coupling in mutant receptors R292A, R310A, K311A, and K314A, we stimulated wild-type or mutant receptors with an Ucn1 concentration (0.5 nM) lower than the concentration threshold required to activate G␣ i proteins (1-10 nM). All mutants exhibited increased agonist-induced cAMP production compared with wild-type receptor responses (Fig. 4b) that were not altered by PTX pretreatment, confirming absence of any significant contribution from R i responses and suggesting involvement primarily of G␣ s proteins (R T ϭ R s response).
We next considered the possibility that altered receptor-G protein coupling efficiency and AC activation in the mutant receptors was due to modification of intracellular mechanisms downstream of receptor-G protein interactions. This was evaluated by assessing Ucn1-induced activation of AC (with or without PTX pretreatment) in cell membranes isolated from HEK293 cells transiently expressing wild-type CRH-R1␣ or mutant receptors. The results of these studies and studies of cAMP accumulation were similar (supplemental Fig. 3), confirming that the mutation-related effects on receptor signaling characteristics involved events near or at the plasma membrane that altered receptor-G protein interactions.
We also assessed the importance of specific amino acids within IC3 in determining the ability of CRH-R1␣ to activate ERK1/2. By measuring Ucn1-induced stimulation of ERK1/2 phosphorylation in HEK293 cells expressing mutant receptors, we found that all mutant receptors had variably reduced ability to transduce agonist activation of ERK1/2. The I293A substitution resulted in the most dramatic effect, as ERK1/2 activation was diminished by 70%; all other mutant receptors exhibited loss of activity by 30 -60% (Fig. 5b), indicating that these amino acid residues are each important for receptor coupling to the ERK1/2 pathway. It is noteworthy that the CRH-R1␣(C-4A) mutant, which is essentially a K311A/K314A double mutant, was significantly more impaired in ERK1/2 responses than either the K311A or K314A mutant alone, suggesting an interaction between the two native amino acids. Because our previous observations suggested that specific amino acids within Arg 292 -Lys 297 and Arg 310 -Lys 314 terminal regions (especially residues Arg 292 , Lys 297 , Arg 310 , and Lys 311 ) as well as Arg 299 , were important for CRH-R1␣ coupling to PTX-sensitive G proteins, we next investigated the role of G i/o proteins in wild-typeand mutant receptor-mediated ERK1/2 activation. Pretreatment of cells expressing wild-type CRH-R1␣ with PTX resulted in a significant attenuation by 50 -60% of maximal ERK1/2 phosphorylation induced by 100 nM Ucn1 (Fig. 6a). This result indicated substantial involvement of G i/o proteins in ERK1/2 activation, in agreement with previous studies (4,25) as well as contribution of other pathways potentially involving PTX-insensitive G proteins and/or G protein-independent mechanisms, as demonstrated previously (26).
We used two complementary approaches to confirm that mutant receptors had altered coupling to G proteins after Ucn1 activation: overexpression of a DN G␣ i (Q205L/D273N) in HEK293 cells and co-immunoprecipitation studies to demonstrate altered receptor G i protein coupling following Ucn1 activation. We found that Ucn1 stimulation of HEK293 cells expressing CRH-R1␣(K314A) and DN G␣ i protein (Q205L/ D273N) had significantly reduced (by 70 -80%) ERK1/2 phosphorylation, which further supported the involvement of G␣ i protein in receptor-mediated response. By contrast, the DN G␣ i protein had no effect on CRH-R1␣(R292A)-mediated ERK1/2 phosphorylation (Fig. 6c). Similarly, after Ucn1 (100 nM) treatment, there was increased co-immunoprecipitation of G␣ i1/2 proteins with wild-type or CRH-R1␣(K314A) receptor proteins (Fig. 6d). Furthermore, when the DN G␣ i protein was co-expressed in HEK293 cells, Ucn1 treatment did not increase the amount of G␣ i protein precipitated. In agreement with our previous observations in membranes from cells expressing CRH-R1␣(R292A), the agonist had a minimal effect on receptor-G i protein association, and only small amounts of G␣ i protein were co-precipitated with the receptor.
CRH-R1␣ IC3 Residues Important for Phospholipase C Activation and IP 3 Production-CRH-R1␣-induced activation of ERK1/2 also involves PTX-insensitive G␣ q -mediated pathways that lead to downstream activation of phospholipase C and intracellular IP 3 accumulation (4). Therefore, we assessed the potential involvement of amino acid residues within receptor IC3 in pathways leading to IP 3 production. In agreement with previous studies (4), treatment of cell membrane suspensions isolated from cells expressing wild-type CRH-R1␣ with Ucn1 (1-250 nM), stimulated a significant increase in IP 3 production by 220 -250%, with a threshold of 10 nM and a maximum response at 100 nM (data not shown). Previous studies (9) suggested that CRH-R1␣-induced IP 3 accumulation involves G␣ qmediated pathways with contribution from PTX-sensitive pathways via G␤␥ dimers released from heterotrimeric G i proteins. Our results supported this because PTX treatment caused a small but significant reduction by 15-20% in Ucn1induced IP 3 production (Fig. 7a). Cell membranes prepared from HEK293 cells co-expressing CRH-R1␣ and DN G␣ q (Q209L/D227N) had significantly reduced (by 89 Ϯ 5%) IP 3 accumulation, thus confirming the involvement of G␣ q proteins in receptor-mediated IP 3 response.
Investigation of the ability of mutant receptors to induce IP 3 production identified that mutation of residues Ile 293 and Lys 314 significantly impaired receptor sensitivity and maximal response, whereas mutation of residues Leu 294 , Met 295 , and Lys 297 reduced maximal response without affecting receptor sensitivity (Table 1). Compared with the wild-type receptor, these mutant receptors exhibited reduced IP 3 production even after PTX pretreatment (Fig. 7b), thus confirming that these mutations mainly modified receptor coupling to PTX-insensitive G proteins involved in IP 3 production, most likely G␣ q protein. All other mutant receptors showed IP 3 production comparable with that of the wild-type receptor.

DISCUSSION
Despite significant advances in understanding the structural determinants that affect binding of CRH-R1 to its cognate agonists CRH and Ucn1, the structural motifs important for determining the ability of CRH-R1 to trigger distinct signaling responses remain poorly defined. Our study focused on the CRH-R1 proximal and distal terminal segments of IC3, because for many GPCRs, the cationic ␣-helices in the juxtamembrane regions near transmembrane domains (TMDs) 5 and 6 appear

TABLE 1 CRH-R1 agonist-induced IP 3 production in HEK293 cells expressing wild-type and alanine mutants of CRH-R1␣
HEK293 cells transfected with wild-type or IC3 mutant CRH-R1␣ receptor cDNAs were assayed for Ucn1-stimulated IP 3 production 72 h after transfection as described under "Experimental Procedures." Cell membrane suspensions (40 g) were prepared and treated with different concentrations of Ucn1 (0 -10 Ϫ6 M) for 30 min at room temperature, followed by the addition of 200 l of IP3 generation buffer and further incubation for 3 min at 37°C. This was followed by extraction of inositol phosphates and neutralization. IP3 levels were determined by competitive binding assay. Results are expressed as the mean Ϯ S.E. of four estimations from three independent experiments. *, p Ͻ 0.05 compared with wild-type CRH-R1␣. to be major functional domains for receptor-G protein coupling and generation of downstream intracellular responses. In particular, the motifs B-B-X-B, or B-X-X-B where B is a basic residue and X is a nonbasic residue, have been identified as important for activation of G proteins and signal transduction as well as receptor maturation and expression (29 -35). This motif, primarily detected in the IC3-TM6 junction, is one of the few structurally conserved elements with established functional role shared between the A and B families of GPCRs. Sequence alignment suggests that all B1 GPCRs family members contain the sequence K/R-A/L-X-K/R (Fig. 7a). Moreover, in 11 of 15 class B1 GPCRs, including the CRH-R1, the TM5-IC3 junction contains the conserved hydrophobic motif B-I/L/ V-L-X, which also appears to be crucial for efficient G protein coupling.

Receptor
Site-specific mutations within the terminal segments of IC3 of the hCRH-R1␣ were constructed and used to probe the role of these domains in coupling to G proteins and signaling pathways that activate the cAMP/PKA and ERK1/2 pathways. One striking finding was that simultaneous disruption of the cationic ␣-helices near TMD 5 and 6 by tandem Ala substitutions, and presumably alteration of the conformation and orientation of IC3, abolished the ability of CRH-R1␣ to transduce signals across the cell, highlighting the important role of IC3 for G protein coupling. The CRH-R1␣(N-4A/C-4A) mutant also exhibited reduced agonist binding affinity which might be due to an alteration of the overall conformation of receptor associated with a disrupted G protein/receptor interaction. Agonist interaction with the G protein-uncoupled CRH-R1 has been shown to weakly stabilize agonist binding, whereas efficient G protein coupling was required for stabilization of agonist-receptor interaction and high affinity binding (36).
Our results also suggest that the cationic ␣-helices Arg 292 -Lys 297 and Arg 310 -Lys 314 contain specific amino acids that are important determinants of CRH-R1␣ coupling to specific G proteins and activation of the AC /cAMP pathway, a crucial signaling event for the activation of the HPA axis and the stress response. The role of these residues in regulating formation of the receptor-G protein ternary complex was evident only in the agonist-activated state, as mutants did not exhibit altered basal (constitutive) activity. Our study showed that within these microdomains, charged amino acids such as Arg 292 , Lys 297 , Arg 310 , Lys 311 , and Lys 314 , as well as hydrophobic amino acids Leu 294 or Met 295 (albeit to a lesser degree) are likely key determinants of the primary receptor recognition sites for G s and G i protein as well as G q protein coupling. Because amino acid substitutions (Leu/Met 3 Ala) of the latter group would abolish hydrophobicity without a significant effect on receptor conformation, this raises the possibility that the primary structure and aliphatic side chain of Leu 294 and Met 295 is important for receptor IC3-G protein interactions. As mentioned above, these cationic ␣-helical domains have been studied extensively in both family A and B GPCRs and in most cases are required for efficient G protein interaction and signal transduction. It has been proposed that for some GPCRs, the helix stabilizes the wall of the G protein pocket by packing against the distal portion of IC3 (37). Surprisingly, we uncovered an unexpected role of these amino acids because disruption of each of these terminal segments enhanced cAMP signaling, suggesting an alternative, not previously described, function of these amino acid clusters that restrain CRH-R1␣ ability to activate the AC/cAMP pathway. The position of charged amino acids within IC3 also appears to be important for determining their contribution to G protein coupling: charged amino acids such as Arg 299 appear to promote efficient receptor IC3-G protein interactions. We showed previously (5) that the ability of the CRH-R1␣ receptor to activate this pathway is determined by the opposing actions of G s and G i proteins, thus the effects of the Ala mutations might be explained in the context of either removal of putative conformational constraints of the secondary structure that optimize the G s protein binding pocket or, alternatively, disruption of the G i protein binding pocket, or both. Indeed, our studies showed that specific amino acids such as Arg 292 , Arg 310 , and Lys 311 are key functional determinants of IC3 interaction with both G s and G i proteins, whereas other amino acids in this region, such as Leu 294 , Met 295 , and Lys 314 exhibit G s protein interaction selectivity (Fig. 8b). Our previous studies (5) have shown that the position of charged amino acid residues within IC3 is critical for receptor G protein coupling: for example introduction of a basic charged amino acid at position 301 within the Ser/Thr amino acid cluster (Ser 301 -Thr 306 ) of IC3 impairs the ability of CRH-R1␣ to couple efficiently to G s proteins, whereas substitution of Ser 301 with an acidic amino acid abolishes G i protein activation.
This differential contribution of specific amino acids depending on the relative position within IC3 could be associated with the ability of the receptor to adopt different conformations. It has been proposed that the CRH-R1 can adopt at least two distinct configurations, which couple to G s or G i protein (40). Ala mutation at the Arg 292 or Lys 314 position might alter receptor-G s protein interaction due to either loss of charge or a hydrophilic residue that helps anchor the receptor at the plasma membrane and thus might allow IC3 to a conformation that unmasks G s protein interaction points or possibly favors interaction with other GPCR-interacting proteins. The importance of basic residues in receptor interactions with interacting protein has been established; for example an arginine cluster within IC3 of the vasopressin V 2 receptor has been identified as the motif important for receptor interaction with GC1q-R (41).
Recent crystallization of the ␤ 2 -adrenergic receptor-G s complex provided the first high resolution insight into the mechanism of signal transduction across the plasma membrane by a GPCR (38,39). These studies identified that movements of TMD5 and 6 that destabilize the receptor inactive state and result in conformational changes of IC3 are crucial steps for ␤ 2 -adrenergic receptor-G s interaction. Therefore, it is possible that the CRH-R1 IC3 amino acids identified in this study play unique roles in this mechanism of complex formation and coupling to distinct G proteins.
An important determinant of CRH-R1␣-G protein interactions that determine the bell-shaped cAMP response appears to be the activating concentration of the agonist. In our studies, subnanomolar Ucn1 concentrations failed to induce receptor-G i protein interactions, and all IC3 mutations tested did not alter these characteristics. Interestingly, comparison of doseresponse curves between mutant and wild-type receptors and responses to PTX treatment identified a complex relationship and suggest that at high agonist concentrations IC3 mutant receptors might exhibit reduced coupling to PTX-sensitive G i/o proteins or alternatively increased activation of PTX-insensitive G proteins that exert an inhibitory action on AC activation (42). Previous studies (9) speculated that a possible receptor dimer formation might induce an allosteric inhibition of AC at high agonist concentrations.
The IC3 terminal segments were also shown to be important for receptor ability to induce ERK1/2 activation. Targeted disruption of the terminal cationic ␣-helices in mutants CRH-R1␣(N-4A) and R1␣(C-4A) led to a significant loss of agonistinduced ERK1/2 responses. Investigating the impact of Ala mutations on specific components of a complex mechanism that involves multiple G protein mediated pathways (4, 5, 28), we identified again Arg 292 , Lys 297 , Arg 299 , Arg 310 , and Lys 311 as critical residues for PTX-sensitive G protein (G i/o )-dependent ERK1/2 activation, and Ile 293 , Leu 294 , Met 295 , Lys 297 , and Lys 314 as important for other pathways involved in ERK1/2 activation. In fact, the hydrophobic microdomain Ile 293 -Met 295 and the basic residues Lys 297 and Lys 314 present in the proximal and distal segments, respectively, appear to be critical structural determinants for the CRH-R1␣-PTX-insensitive G␣ q protein interaction that mediates phospholipase C activation and intracellular IP 3 accumulation. Basic residues within the juxtamembrane regions of IC3 have been shown to permit efficient coupling to G q also for other class A and B GPCRs (15,43).
Recently, a polybasic motif in the C terminus of M3R was identified as critical for preassembly of M3 muscarinic acetylcholine receptors inactive state complexes with G q , an interaction that increases the sensitivity and accelerates the onset of signaling (44).
In conclusion, the juxtamembrane regions of CRH-R1␣ contain microdomains crucial for receptor ability to differentially recognize and couple to distinct G proteins and downstream signaling. In particular, charged amino acids at either proximal or distal terminal segments play key roles for limiting G s coupling and for activation of G i proteins and thus provide a tight control of CRH-R1 signaling to prevent excess activity and hyperstimulation of the "stress" axis that might lead to pathological conditions.