Motif Mutation of Bradykinin B2 Receptor Second Intracellular Loop and Proximal C Terminus Is Critical for Signal Transduction, Internalization, and Resensitization
1998; Elsevier BV; Volume: 273; Issue: 50 Linguagem: Inglês
10.1074/jbc.273.50.33548
ISSN1083-351X
AutoresGregory N. Prado, Dale F. Mierke, Maria Pellegrini, Linda Taylor, Péter Polgàr,
Tópico(s)Peptidase Inhibition and Analysis
ResumoIn the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638–14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a K d of approximately 1.1 nm. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization. In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638–14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a K d of approximately 1.1 nm. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization. The bradykinin B2 receptor (BKB2R) 1The abbreviations used are: BKB2R, bradykinin B2 receptor; BKB2, bradykinin B2; GPCR, G protein-coupled receptor; AT1, angiotensin II type 1; MD, molecular dynamics; WT, wild type; ARA, arachidonic acid; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; IC, intracellular loop; TM, transmembrane. 1The abbreviations used are: BKB2R, bradykinin B2 receptor; BKB2, bradykinin B2; GPCR, G protein-coupled receptor; AT1, angiotensin II type 1; MD, molecular dynamics; WT, wild type; ARA, arachidonic acid; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; IC, intracellular loop; TM, transmembrane. cDNA has been successfully transfected into such cell types as hamster lung fibroblasts, Rat-1 fibroblasts and CHO cells with the expected binding properties (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 2Taylor L. Ricupero D. Jean J.C. Jackson B.A. Polgar P. Biochem. Biophys. Res. Commun. 1992; 188: 786-793Crossref PubMed Scopus (20) Google Scholar, 3Eggerickx D. Raspe E. Bertrand D. Vassart G. Parmentier M. Biochem. Biophys. Res. Commun. 1992; 187: 1306-1313Crossref PubMed Scopus (111) Google Scholar). The BKB2R is a typical G protein-coupled receptor (GPCR) that has been reported to be associated with Gq (4Gutowski S. Smrcka A. Nowak L. Wu D. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 5Ricupero D.A. Polgar P. Sowell M.O. Gao Y. Baldwin G. Taylor L. Mortensen R.M. Biochem. J. 1997; 327: 1-7Crossref PubMed Scopus (12) Google Scholar, 6Yanaga F. Hirata M. Koga T. Biochim. Biophys. Acta. 1991; 1094: 139-146Crossref PubMed Scopus (38) Google Scholar, 7Wolsing D.H. Rosenbaum J.S. J. Pharmacol. Exp. Ther. 1993; 266: 253-261PubMed Google Scholar), Gi, and G12 (8Wilk-Blaszczak M.A. Singer W.D. Belardetti F. J. Neurophysiol. 1996; 76: 3559-3562Crossref PubMed Scopus (7) Google Scholar, 9Liao J.K. Homcy C.J. J. Clin. Invest. 1993; 92: 2168-2172Crossref PubMed Scopus (107) Google Scholar, 10Miyamoto A. Laufs U. Pardo C. Liao J.K. J. Biol. Chem. 1997; 272: 19601-19608Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Binding of a GPCR to more than one Gα subunit is not unprecedented. For example, the angiotensin II type 1 (AT1) receptor couples to both Gi and Gq (11Shibata T. Suzuki C. Ohnishi J. Murakami K. Miyazaki H. Biochem. Biophys. Res. Commun. 1996; 218: 383-389Crossref PubMed Scopus (57) Google Scholar). Whereas Gq has been linked to phospholipase C activation (6Yanaga F. Hirata M. Koga T. Biochim. Biophys. Acta. 1991; 1094: 139-146Crossref PubMed Scopus (38) Google Scholar, 7Wolsing D.H. Rosenbaum J.S. J. Pharmacol. Exp. Ther. 1993; 266: 253-261PubMed Google Scholar), the release of arachidonate, via phospholipase A2 activation, has been linked to Gi and Gs (12Ricupero D. Taylor L. Polgar P. Agents Actions. 1993; 40: 110-118Crossref PubMed Scopus (20) Google Scholar, 13Pyne N.J. Tolan D. Pyne S. Biochem. J. 1997; 328: 689-694Crossref PubMed Scopus (44) Google Scholar, 14Castano M.E. Schanstra J.P. Hirtz C. Pesquero J.B. Pecher C. Girolami J.P. Bascands J.L. Am. J. Physiol. 1998; 274: F532-F540PubMed Google Scholar).The cellular response of GPCRs to agonists is regulated under a tightly controlled process of desensitization and resensitization (15Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar, 16Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar). Desensitization prevents cells from uncontrolled stimulation, and resensitization allows cells to recover and maintain responsiveness (17Bohm S.K. Khitin L.M. Smeekens S.P. Grady E.F. Payan D.G. Bunnett N.W. J. Biol. Chem. 1997; 272: 2363-2372Crossref PubMed Scopus (71) Google Scholar). Both processes appear to be regulated at the receptor level. Recent studies by Haasemann et al. (18Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar) showed that stimulation of BKB2R results in redistribution of the receptor and its internalization in caveolae (18Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar). On/off regulatory elements have been identified in other receptors (19Nussenveig D.R. Heinflink M. Gershengorn M.C. J. Biol. Chem. 1993; 268: 2389-2392Abstract Full Text PDF PubMed Google Scholar, 20Thomas W.G. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Crossref PubMed Scopus (110) Google Scholar, 21Benya R.V. Fathi Z. Battey J.F. Jensen R.T. J. Biol. Chem. 1993; 268: 20285-20290Abstract Full Text PDF PubMed Google Scholar, 22Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, to date, no clear consensus motifs have been identified to determine either internalization or resensitization. The DRYXXI/VXXP motif located in the second intracellular loop (IC2) of the human muscarinic cholinergic receptor has been proposed as important for receptor internalization (23Moro O. Lameh J. Högger P. Sadée W. J. Biol. Chem. 1993; 268: 22273-22276Abstract Full Text PDF PubMed Google Scholar). Another important motif is the tyrosine-containing motif located in the proximal BKB2R C terminus. The motifs in this region have been implicated in receptor internalization in other GPCRs, such as neurokinin 1 receptor (17Bohm S.K. Khitin L.M. Smeekens S.P. Grady E.F. Payan D.G. Bunnett N.W. J. Biol. Chem. 1997; 272: 2363-2372Crossref PubMed Scopus (71) Google Scholar), parathyroid hormone receptor (24Huang Z. Chen Y. Nissenson R.A. J. Biol. Chem. 1995; 270: 151-156Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and AT1 receptor (20Thomas W.G. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Crossref PubMed Scopus (110) Google Scholar). Studies have also shown that clathrin-associated protein complexes interact with tyrosine-based motifs (25Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacio J.S Science. 1995; 209: 1872-1875Crossref Scopus (817) Google Scholar).Mutations exchanging single as well as multiple amino acids have been applied to resolve the actions of GPCRs. The importance of specific residues within the IC face of GPCRs was demonstrated recently by a number of investigators, using receptors such as BKB2 receptors (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), AT1 receptors (26Conchon S. Barrault M.B. Miserey S. Corvol P. Clauser E. J. Biol. Chem. 1997; 272: 25566-25572Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), interleukin-8 receptors (27Xie W. Jiang H. Wu Y. Wu D. J. Biol. Chem. 1997; 272: 24948-24951Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and muscarinic acetylcholine receptors (28Lee N.H. Geoghagen N.S. Cheng E. Cline R.T. Fraser C.M Mol. Pharmacol. 1996; 50: 140-148PubMed Google Scholar). Our previous results showed that Tyr131 and Tyr322 are crucial for signal transduction (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Truncation of the distal C terminus of BKB2R exhibited normal BK-activated ARA release, PI turnover, and [Ca2+]i flux. Furthermore, our previous results showed that at least some of the structural elements involved in the internalization process are found in the IC2 and the proximal as well as distal C terminus (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Reports of the use of models of BKB2R to explore ligand binding have appeared in the literature (29Kyle D.J. Chakravarty S. Sinsko J.A. Stormann T.M. J. Med Chem. 1994; 37: 1347-1354Crossref PubMed Scopus (80) Google Scholar, 30Doughty S.W. Reynolds C.A Biochem. Soc. Trans. 1996; 24: 259-263Crossref PubMed Scopus (2) Google Scholar, 31Jarnagin K. Bhakta S. Zuppan P. Yee C. Ho T. Phan T. Tahilramani R. Pease J.H.B. Miller A. Freedman R. J. Biol. Chem. 1996; 271: 28277-28286Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). For example, Kyle et al. (29Kyle D.J. Chakravarty S. Sinsko J.A. Stormann T.M. J. Med Chem. 1994; 37: 1347-1354Crossref PubMed Scopus (80) Google Scholar) proposed that ligand binding to the receptor is in a twisted S shape with the C-terminal β-turn lodged inside the receptor. Doughty and Reynolds (30Doughty S.W. Reynolds C.A Biochem. Soc. Trans. 1996; 24: 259-263Crossref PubMed Scopus (2) Google Scholar) generated a BKB2R model showing an interaction between TM6 and TM7 through an Arg281/Asp266 attraction. However, at this time, the conformational features of the intracellular loops and termini of the BKB2R have not been reported.Herein, we further investigate the relationship between Tyr131 and Tyr322 in terms of signaling, uptake, and resensitization. In conjunction with our results, we generated a molecular model of BKB2R with these two regions in close spatial proximity. We also investigate the role of an apparently critical threonine at position 137 in IC2. Alignment of GPCRs of the peptide subfamily shows that proline is generally conserved at this position. However, the position occupied by this proline is a tryptophan in endothelin receptors and a serine in galanin receptors. Our results suggest that threonine 137 participates in the regulation of BKB2R internalization and resensitization, whereas BKB2R desensitization does not involve this residue.DISCUSSIONWe have generated a number of mutants within the IC2 and proximal C terminus of the BKB2R. All constructs retained the binding kinetics of the WT receptor. However, the mutations showed marked and varied effects on BKB2R signal transduction, receptor internalization, and resensitization. These results suggest that no one motif within the IC face of the BKB2R is solely responsible for a given receptor function. Instead, cooperative interactions appear to be taking place among the various IC domains.For example, replacement of Tyr322, located at the proximal C terminus, with the small, uncharged alanine had little effect on either BK-stimulated ARA release or PI turnover. However, replacement with the small, hydroxyl-containing serine reduced both events markedly. Replacement of Tyr131 with serine also markedly reduced both events. However, when both tyrosine residues, Tyr131 and Tyr322, were mutated to serine (YSYS), the consequent reduction of a bulky residue only impaired the activation of phospholipase C. The double mutation did not affect receptor-stimulated ARA release. In contrast, double mutation of Tyr131 and Tyr322 residues to alanine (YAYA) impaired ARA release but not PI turnover. In this case, a hydrophobic residue at both locations promotes phospholipase C action but hinders the release of ARA. However, a double substitution with phenylalanine, a bulky hydrophobic residue, hinders both signaling pathways. These results demonstrate that discrete sequences within the DRY motif of IC2 and the tyrosine-based motif in the proximal C terminus interact with each other and cause differential effects on BK-activated signal pathways. A possible explanation for this differential signaling is that Tyr322 of the BKB2R is phosphorylated and acts like the YIPP motif, similarly found in the proximal C tail of the AT1 receptor (41Venema R.C. Ju H. Venema V.J. Schieffer B. Harp J.B. Ling B.N. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 7703-7708Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, our results and results reported by Blaukatet al. (42Blaukat A. Alla S.A. Lohse M.J. Müller-Esterl W. J. Biol. Chem. 1996; 271: 32366-32374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) indicate that this tyrosine residue is not phosphorylated. A more solid explanation is that the two IC regions are cooperatively involved in coupling to more than a single subset of G protein (Gq, Gi, or Gi-like proteins). Interaction between the IC loops and the proximal C tail has been proposed in other GPCRs (43Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar). In the β2-adrenergic receptor, the C-terminal portion of IC3 and the proximal C tail are important for Gs activation (44O'Dowd B.F. Hnatowich M. Regan J.W. Leader W.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 15985-15992Abstract Full Text PDF PubMed Google Scholar). In the BKB2R, a small hydrophobic residue such as alanine at Tyr131 and Tyr322 favors interaction with a G protein responsible for PI turnover (Gq). In all, residue size, hydrophobicity, and charge at these positions play important roles in signal transduction. Another example to illustrate the effect of charge is the substitution of the hydrophilic threonine residue at location 137 with either proline or aspartate. Exchange of threonine for proline had no effect on either ARA release or PI turnover. However, the T137D mutant receptor, which displays a negative charge, showed impaired PI turnover and ARA release. Our previous studies demonstrated that point mutations at Tyr131 to serine, alanine, or phenylalanine also affected the rate of receptor internalization as compared with that of WT (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Y131A displayed the slowest rate of uptake, whereas Y131S displayed a rate considerably faster than WT. Our present results further demonstrate that the double mutant YAYA also displayed a very slow rate of uptake, notably slower than WT or the other double mutants. These results confirm the importance of the DRY region in receptor internalization. Apparently, mutant receptors that display either the slowest or the most rapid rates of receptor internalization still show a degree of PI turnover similar to that of WT receptor. This suggests that signal transduction in relation to the Tyr131/Tyr322 interaction is not involved directly with receptor uptake. A similar lack of correlation between internalization and signal transduction was observed in muscarinic (18Haasemann M. Cartaud J. Muller-Esterl W. Dunia I. J. Cell Sci. 1998; 111: 917-928Crossref PubMed Google Scholar), angiotensin/AT1 (45Hunyady L. Bor M. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1995; 270: 16602-16609Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and β2-adrenergic receptors (46Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar).To further understand the role of IC2 in receptor uptake, we investigated the DRYXXI/VXXPφ motif as proposed in muscarinic cholinergic receptor (23Moro O. Lameh J. Högger P. Sadée W. J. Biol. Chem. 1993; 268: 22273-22276Abstract Full Text PDF PubMed Google Scholar). We aligned this motif with 184 GPCRs belonging to the peptide subfamily. We found that proline is conserved in 71% of the receptors. Conversion of threonine in the BKB2R to proline resulted in a complete loss of receptor uptake. However, substitution of proline at this site did not affect signal transduction. Substitution with a negatively charged aspartate to generate T137D reversed these effects. T137D did not stimulate ARA release or PI turnover but returned receptor uptake to that of WT. This result points to the importance of a negative charge at this site and suggests that phosphorylation at Thr137 participates in termination of receptor function and initiation of receptor uptake. Blaukat et al. (42Blaukat A. Alla S.A. Lohse M.J. Müller-Esterl W. J. Biol. Chem. 1996; 271: 32366-32374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) demonstrated threonine/serine phosphorylation to be taking place in conjunction with BKB2R internalization and resensitization.Our results further suggest that internalization and resensitization are linked, whereas desensitization is a more complicated process related to specific metabolic events regulated by BK. When resensitization was tested by measuring [Ca2+]i, T137P, which was only minimally taken up even after 60 min, showed no resensitization. On the other hand, YFYF, with a moderate rate of internalization, resensitized similarly to WT. YAYA, which was also taken up poorly, but not to the extreme of T137P, resensitized somewhere between YFYF and T137P. It remains to be resolved which receptor motif(s) determine internalization, which determine desensitization, and what correlation exists between these two actions.Our present and previous results, in sum, clearly indicate cooperativity or interaction between the C-terminal tail and IC2 of the receptor. We postulate the presence of α-helices (for the C terminus) and a β-turn (residues 137–138 of IC2) based on homology analysis with structurally defined proteins, following a procedure recently reported for the κ-opiate receptor (36Paterlini G. Portoghese P.S. Ferguson D.M. J. Med. Chem. 1997; 40: 3254-3262Crossref PubMed Scopus (74) Google Scholar). However, the relative orientation of the loops and termini, containing the α-helices or β-turns, needs to be established. The aim of our modeling effort is to investigate the conformations with these two regions (IC2 and C terminus) in close proximity. Given the constraints (i.e.presence of the α-helices and β-turn), we looked for conformations that are energetically feasible. There are three possible orientations that would place the C-terminal helix (residues 310–329) in close proximity to Tyr131: it could lie 1) on the membrane surface near TM6-TM5, 2) on the membrane surface near TM1-TM2, or (3Eggerickx D. Raspe E. Bertrand D. Vassart G. Parmentier M. Biochem. Biophys. Res. Commun. 1992; 187: 1306-1313Crossref PubMed Scopus (111) Google Scholar) across the cytoplasmic ends of the helix bundle. All attempts at placing the C-terminal helix toward TM1-TM2 failed. The topological orientation of the helical bundle does not allow for Tyr131and Tyr322 to come close without disrupting the C-terminal helix or the TM bundle. In contrast, during the simulations starting with the other two orientations, low energy conformations were adapted early and maintained for the remainder of the 200 ps of MD simulation. Of these two, we prefer the first, in which the C-terminal helix lies on the membrane surface, mimicked by a layer of decane molecules in the simulation, near TM6-TM5. In the resulting structure, the hydrophobic face of the amphipathic C-terminal helix projects into the membrane surface. This conformation also places Cys326, postulated to be a site of palmitoylation, adjacent to the membrane surface. One role of the palmitoylation of this residue would be to securely anchor this helix to the membrane surface. Based on this, we exclude the low energy conformations that place the α-helix of the C terminus across the helix bundle, even though from an energetic perspective based on the MD simulations, it is feasible. In Fig. 9, two views of the resulting conformation from the MD simulation, placing Tyr131 and Tyr322 8.1 Å distal from each other, are shown. The resulting conformation places IC3 juxtaposed to IC2 and the C terminus. This is important given the well documented role of IC3 in coupling to the G proteins.Concerning Thr137 and the β-turn, the resulting conformations have this turn well exposed, projecting down into the solvent, readily available for phosphorylation. Mutations at this position indicate that following receptor activation, leading to signal transduction, phosphorylation at this position results in termination of the activated receptor structure. This termination is then followed by internalization and potential resensitization of the receptor. A model of the T137P mutation suggests that it stabilizes the β-turn structure, while removing the possibility of phosphorylation.In summary, our results using BKB2R mutant receptors and consequent molecular modeling involving the distal IC2 and proximal C terminus point to important interaction between these two regions, not only with regard to regulation of signal transduction but also in conjunction with receptor internalization and resensitization. Apparently, the motifs encompassing Tyr131 and Tyr322, perhaps in conjunction with elements within the IC3, dictate BKB2R-G protein interaction. The bradykinin B2 receptor (BKB2R) 1The abbreviations used are: BKB2R, bradykinin B2 receptor; BKB2, bradykinin B2; GPCR, G protein-coupled receptor; AT1, angiotensin II type 1; MD, molecular dynamics; WT, wild type; ARA, arachidonic acid; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; IC, intracellular loop; TM, transmembrane. 1The abbreviations used are: BKB2R, bradykinin B2 receptor; BKB2, bradykinin B2; GPCR, G protein-coupled receptor; AT1, angiotensin II type 1; MD, molecular dynamics; WT, wild type; ARA, arachidonic acid; PI, phosphoinositide; DMEM, Dulbecco's modified Eagle's medium; IC, intracellular loop; TM, transmembrane. cDNA has been successfully transfected into such cell types as hamster lung fibroblasts, Rat-1 fibroblasts and CHO cells with the expected binding properties (1Prado G.N. Taylor L. Polgar P. J. Biol. Chem. 1997; 272: 14638-14642Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 2Taylor L. Ricupero D. Jean J.C. Jackson B.A. Polgar P. Biochem. Biophys. Res. Commun. 1992; 188: 786-793Crossref PubMed Scopus (20) Google Scholar, 3Eggerickx D. Raspe E. Bertrand D. Vassart G. Parmentier M. Biochem. Biophys. Res. Commun. 1992; 187: 1306-1313Crossref PubMed Scopus (111) Google Scholar). The BKB2R is a typical G protein-coupled receptor (GPCR) that has been reported to be associated with Gq (4Gutowski S. Smrcka A. Nowak L. Wu D. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 5Ricupero D.A. Polgar P. Sowell M.O. Gao Y. Baldwin G. Taylor L. Mortensen R.M. Biochem. J. 1997; 327: 1-7Crossref PubMed Scopus (12) Google Scholar, 6Yanaga F. Hirata M. Koga T. Biochim. Biophys. Acta. 1991; 1094: 139-146Crossref PubMed Scopus (38) Google Scholar, 7Wolsing D.H. Rosenbaum J.S. J. Pharmacol. Exp. Ther. 1993; 266: 253-261PubMed Google Scholar), Gi, and G12 (8Wilk-Blaszczak M.A. Singer W.D. Belardetti F. J. Neurophysiol. 1996; 76: 3559-3562Crossref PubMed Scopus (7) Google Scholar, 9Liao J.K. Homcy C.J. J. Clin. Invest. 1993; 92: 2168-2172Crossref PubMed Scopus (107) Google Scholar, 10Miyamoto A. Laufs U. Pardo C. Liao J.K. J. Biol. Chem. 1997; 272: 19601-19608Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Binding of a GPCR to more than one Gα subunit is not unprecedented. For example, the angiotensin II type 1 (AT1) receptor couples to both Gi and Gq (11Shibata T. Suzuki C. Ohnishi J. Murakami K. Miyazaki H. Biochem. Biophys. Res. Commun. 1996; 218: 383-389Crossref PubMed Scopus (57) Google Scholar). Whereas Gq has been linked to phospholipase C activation (6Yanaga F. Hirata M. Koga T. Biochim. Biophys. Acta. 1991; 1094: 139-146Crossref PubMed Scopus (38) Google Scholar, 7Wolsing D.H. Rosenbaum J.S. J. Pharmacol. Exp. Ther. 1993; 266: 253-261PubMed Google Scholar), the release of arachidonate, via phospholipase A2 activation, has been linked to Gi and Gs (12Ricupero D. Taylor L. Polgar P. Agents Actions. 1993; 40: 110-118Crossref PubMed Scopus (20) Google Scholar, 13Pyne N.J. Tolan D. Pyne S. Biochem. J. 1997; 328: 689-694Crossref PubMed Scopus (44) Google Scholar, 14Castano M.E. Schanstra J.P. Hirtz C. Pesquero J.B. Pecher C. Girolami J.P. Bascands J.L. Am. J. Physiol. 1998; 274: F532-F540PubMed Google Scholar). The cellular response of GPCRs to agonists is regulated under a tightly controlled process of desensitization and resensitization (15Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar, 16Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar). 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