Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling

Abstract

Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism.Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation. Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism. Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation. INTRODUCTIONThe physiological actions of the vasoactive octapeptide hormone angiotensin II (AngII) in the cardiovascular, endocrine, and neuronal systems are mediated by membrane-bond receptors. Two pharmacologically distinct AngII receptors have been identified: AT1 and AT2(1.Chiu A.T. Herblin W.F. McCall D.E. Ardecky R.J. Carini D.J. Duncia J.V. Pease L.J. Wong P.C. Wexler R.R. Johnson A.L. Timmermans P.B.M.W.M. Biochem. Biophys. Res. Commun. 1989; 165: 196-203Crossref PubMed Scopus (836) Google Scholar, 2.Whitebread S. Mele M. Kamber B. De Gasparo M. Biochem. Biophys. Res. Commun. 1989; 163: 284-291Crossref PubMed Scopus (758) Google Scholar). AT1 receptors bind biphenylimidazole antagonists such as DuP753 with high affinity and pentapeptide analogs such as CGP42112A with low affinity, whereas AT2 receptors have the reverse affinities for these compounds. Cloning of both receptor types has revealed that they belong to the seven transmembrane domain receptor family(3.Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1166) Google Scholar, 4.Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-233Crossref PubMed Scopus (773) Google Scholar, 5.Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Abstract Full Text PDF PubMed Google Scholar, 6.Mukoyama M. Nakajima M. Horiuchi M. Sasamura H. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Abstract Full Text PDF PubMed Google Scholar). Two closely related AT1 isoforms (AT1A and AT1B) have been identified in rat and mouse species(7.Iwai N. Inagami T. FEBS Lett. 1992; 298: 257-260Crossref PubMed Scopus (381) Google Scholar, 8.Sasamura H. Hein L. Krieger J.E. Pratt R.E. Kobilka B.K. Dzau V.J. Biochem. Biophys. Res. Commun. 1992; 185: 253-259Crossref PubMed Scopus (279) Google Scholar). AT1 receptors have been shown to be coupled to G-proteins and to activate phospholipase C (PLC)(9.Bottari S.P. de Gasparo M. Steckeling U.M. Levens N.R. Frontiers Neuroendocrinol. 1993; 14: 123-171Crossref PubMed Scopus (338) Google Scholar, 10.Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar). This results in inositol trisphosphate (IP3) generation, which then causes an increase in intracellular calcium concentrations, and diacylglycerol formation, which leads to protein kinase C activation(11.Farese R.V. Larson R.E. Davis J.S. Endocrinology. 1984; 114: 302-304Crossref PubMed Scopus (63) Google Scholar, 12.Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3297) Google Scholar).The molecular location of the ligand binding domain of the G-protein-coupled receptors has been intensively investigated using genetic, biochemical, and biophysical approaches. The binding site for small molecules such as the bioamine neurotransmitters involves polar residues of the transmembrane domains (TM)(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). In contrast, the binding site for large hormones such as the pituitary glycoproteins is located in the large amino-terminal extracellular domain of the corresponding receptors(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The ligand binding domains of peptide receptors, such as AT1, have been more recently investigated. Peptide binding by AT1 receptors is dependent on the presence of four extracellular cysteines (14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar) as well as on several additional residues located in the extracellular domains(15.Hjorth S.A. Schambye H.T. Greenlee W.J. Schwartz T.W. J. Biol. Chem. 1994; 269: 30953-30959Abstract Full Text PDF PubMed Google Scholar). Whereas the binding of non-peptidic ligands is unaffected by these extracellular mutations, numerous polar residues of the hydrophobic transmembrane segments are determinant in binding of non-peptidic antagonists(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 17.Ji H. Leung M. Zhang Y. Catt K.J. Sandberg K. J. Biol. Chem. 1994; 269: 16533-16536Abstract Full Text PDF PubMed Google Scholar, 18.Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar, 19.Schambye H.T. Hjorth S.A. Bergsma D.J. Sathe G. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7046-7050Crossref PubMed Scopus (116) Google Scholar). Two of these residues, Asp74 and Tyr292, also play an essential role in the coupling of AT1A to PLC(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 18.Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar).Biochemical analysis (20.Desarnaud F. Marie J. Lombard C. Larguier R. Seyer R. Lorca T. Jard S. Bonnafous J.C. Biochem. J. 1993; 289: 289-297Crossref PubMed Scopus (27) Google Scholar) and molecular modeling studies (21.Underwood D.J. Strader C.D. Rivero R. Patchett A.A. Greenlee W. Prendergast K. Chem. Biol. 1994; 1: 211-221Abstract Full Text PDF PubMed Scopus (65) Google Scholar) 2P. Broto, unpublished results. indicate a major role for the third and fifth transmembrane segments (TM-III and TM-V) in AngII binding. To define the functional roles of polar residues in these transmembrane domains, of different polar residues Ala and were in the rat AT1A receptor The of the receptors for peptidic and non-peptidic and as well as signaling were of the mutants were for the binding of AngII and analogs. the were located in different domains of the AT1A investigated the recently of be for this peptide hormone This was for different mutants of the receptors as well of the and receptors they were that molecular was transmembrane domains of two different B.K. Kobilka K. R.J. PubMed Scopus Google Scholar, R. J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R. J. FEBS Lett. 1993; PubMed Scopus Google Scholar). The ligand binding and signaling of four binding AT1A mutants were in COS in different and for the receptors were two types of using the rat AT1A in or a which been S. Monnot C. C. Corvol P. Clauser E. Biochem. Biophys. Res. Commun. 1994; PubMed Scopus Google and were in the were on a for of for of for of the for the extracellular was as in an in using the a with a functional the were by of a and with an The mutant was by the corresponding to the amino-terminal extracellular of AT1A and with corresponding to and mutants were using four corresponding to both Ser109 and a site to a site for Asn111 as well as a These were in the Ser105, and and of the AT1A mutants were using the These were in to the amino of the AT1A and were as a to the absence of homologous recombination two AT1A The two corresponding to the and in the and of the To the the the of was the of were by using 2 was as C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar). the AT1A cDNAs were the L. Clauser E. D. M. W.J. 45: Full Text PDF PubMed Scopus Google in and was in with and Two were or with of by the M. C. P. L. Clauser E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). studies or inositol were were in with and The has been previously C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, B. C. Monnot C. Bernstein K.E. T.J. Alexander R.W. Corvol P. Clauser E. Biochem. Biophys. Res. Commun. 1992; 187: PubMed Scopus Google Scholar). To the and were with of the corresponding and 2 of the using the calcium P. J. 1: Google Scholar). were by to and by was by the 1 was by high and were and binding assays were as S. Monnot C. Teutsch B. Corvol P. Clauser E. FEBS Lett. 1994; PubMed Scopus Google Scholar). binding were using and to of the ligands. was in were with a D. Biochem. PubMed Scopus Google of in to of AngII was as Y. de M. M. J. J. J. 1989; PubMed Scopus Google Scholar). were in and with 2 for and then with AngII for in presence of on a the was for of was with or using the P. N. Biochem. PubMed Scopus Google Scholar). was with to were with reverse using of a corresponding to the of the This reverse of the to the and In these and cDNAs be of the was with and then using 1 of and of in a of the were using 1 The for to the 1 2 and are in The and are to the and and the and are to the by of the the two The two to the and the to the AT1A of the and are by were on COS with or or with and The is a absence of due to reverse of cDNAs the analysis was with a or in AT1A the roles of several polar residues in and in the binding of both peptidic and non-peptidic a of rat AT1A receptors were These mutant receptors were expressed in and by binding mutants and are to bind the peptidic and or the non-peptidic not These that these in a of the for peptide and binding or that they are not expressed the that of these two receptor mutants results in normal ligand binding. is that these mutants are expressed the and that Lys102 and Lys199 are essential for the AT1A binding site. Two studies also the role of Lys102 and Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar, S.A. Schambye H.T. Greenlee W.J. Schwartz T.W. J. Biol. Chem. 1994; 269: 30953-30959Abstract Full Text PDF PubMed Google Scholar). In the of was that an in receptor in of the that of this does not a in receptor as protein to receptors, functional in binding that Lys102 an in binding site for peptidic and and non-peptidic ligands be for of this with causes a major in affinity for AngII (14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar) and with Ala the of AngII and DuP753 that Lys199 is for with of AngII as well as with the of the of 1 in a mutants in the and with to of the receptor The of these mutants for different peptidic or non-peptidic ligands was then of these mutants AngII and binding affinities that not of the are in the binding affinity of the CGP42112A or non-peptidic DuP753 for AT2 and AT1 receptors, for the and a increase in affinity for CGP42112A and a in affinity for an affinity for the DuP753 to the but a affinity for CGP42112A 2 in a These results indicate that the polar residues Ser105, Ser107, and Ser109 are not essential for the binding of peptidic or non-peptidic ligand to the AT1A The absence of a major role for in the binding of and DuP753 has been H. Leung M. Zhang Y. Catt K.J. Sandberg K. J. Biol. Chem. 1994; 269: 16533-16536Abstract Full Text PDF PubMed Google Scholar). This is in AT1 receptors, but is by an in the AngII that is not for the for the which peptidic but not non-peptidic results that this is not a to the binding of peptidic or of the non-peptidic ligands. In contrast, these studies the of the residues Asn111 and Ser115 in binding the ligands. These results be related to two the of in by an C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar) and the of the Tyr292, in by a J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar) a affinity for DuP753 and a affinity for on different transmembrane domains, these residues are probably a small of in the that and belong to a ligand binding site. modeling studies that these residues in a that is or four the and in the in G-protein and of binding to the AT1 receptor leads to the activation of which a and two and the of AT1 coupling and signaling be by the of or in to of is that the of the to is dependent on the of binding the of the and that the is with an to the of the M. E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). the coupling of different AT1A mutants be and using the and the these coupling of and mutant receptors was with that of the AT1A in cells. was in not whereas a of was in the the and The of these different AT1A receptors was with AngII and which are in a to the corresponding of AngII for these receptors in a The of is to the receptor for and and to a increase the of These results a functional coupling for these two In contrast, the of by the and mutants was increase the but these receptors were expressed in the or mutants to in the was also by or for the and mutants as to the receptor the and the were for the two mutants as to the receptor These results indicate that the and do not alter the ability of the receptor to to but probably with the of of inositol in COS or mutant AT1A receptors. were for the and AT1A receptors. The results are expressed as the of the to to the of in of Ser115 results in a of the ability of the receptor to formation, the that this receptor is expressed to of the receptor in this polar in a role in activation of the AT1A for G-protein coupling and Two polar residues and located in the and have also been shown to be essential for receptor activation and C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 18.Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar). to a for AT1A receptor Marie have that the of Asp74 and the of are by an analysis of the polar residues the seven transmembrane segments by modeling the of an for the role of these residues and in receptor In the absence of and Asp74 be by an This be by binding of AngII or in the of a and for the receptor activation and G-protein This to be of different AT1A investigated the of AT1A receptors by has been that the transmembrane domains of such as G-protein-coupled receptors, are and in the and in a to B.K. Kobilka K. R.J. PubMed Scopus Google Scholar). of indicate that of this functional does not the transmembrane domains of a but be using of receptors, or two domains two but molecules of B.K. Kobilka K. R.J. PubMed Scopus Google Scholar, R. J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R. J. FEBS Lett. 1993; PubMed Scopus Google Scholar). of such functional G-protein-coupled receptors, of two was recently by of deficient and receptors. In this of of binding or coupling deficient receptors with in on different transmembrane domains or with receptors in restoration of the ligand binding domain or the coupling mechanism. These that functional receptor be by protein the the and two mutants and are to bind the peptidic and or the non-peptidic not Co-expression of four AT1A mutants in different was to be for this peptide hormone with the two mutants not binding for not In contrast, of with in the of binding for the ligand binding to of the receptor in of and for different ligands the of binding with on the in a in a of of on receptor the two in which is in and of results that the two mutants and are expressed the and are to whereas individual in does not in functional receptors. The of binding is to the increase of the of the two that the of these binding the presence of the two deficient mutants in This protein results in the restoration of a binding site to that of the with peptidic and antagonists as well as with non-peptidic compounds. In contrast, was restoration of a ligand binding site the two mutants and were or with the or mutants not of the mutants not in activation of not was were with mutant receptors and that are to of both have been shown to bind AngII with affinities C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar) and the of the and of these was to that homologous recombination the This was using AT1A additional the or or both of the these normal AT1A receptors not and of these in COS the analysis of the by using of located in the or or in the or of the AT1A 2 and with 1 and are the COS with the but not with COS with the and homologous recombination were of two The that functional receptor was the two mutants and were is that homologous recombination does not results that AT1A receptors be to interact with to binding to of the These binding for to of the receptor that the of mutant receptors that the is the as the AT1A in the The of these receptors to a suggests that the of functional molecules is for the to be or that the of these molecules is not for G-protein these that de of an AngII binding receptor two mutants is to transmembrane domains the the identified a role for residues located in the of and and in AngII binding. these residues and in and are involved in the binding site of non-peptidic analogs. In addition to the and Asp74 residues, Ser115 is for receptor Finally, of these different AT1A mutants has permitted the of peptide hormone receptors. INTRODUCTIONThe physiological actions of the vasoactive octapeptide hormone angiotensin II (AngII) in the cardiovascular, endocrine, and neuronal systems are mediated by membrane-bond receptors. Two pharmacologically distinct AngII receptors have been identified: AT1 and AT2(1.Chiu A.T. Herblin W.F. McCall D.E. Ardecky R.J. Carini D.J. Duncia J.V. Pease L.J. Wong P.C. Wexler R.R. Johnson A.L. Timmermans P.B.M.W.M. Biochem. Biophys. Res. Commun. 1989; 165: 196-203Crossref PubMed Scopus (836) Google Scholar, 2.Whitebread S. Mele M. Kamber B. De Gasparo M. Biochem. Biophys. Res. Commun. 1989; 163: 284-291Crossref PubMed Scopus (758) Google Scholar). AT1 receptors bind biphenylimidazole antagonists such as DuP753 with high affinity and pentapeptide analogs such as CGP42112A with low affinity, whereas AT2 receptors have the reverse affinities for these compounds. Cloning of both receptor types has revealed that they belong to the seven transmembrane domain receptor family(3.Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1166) Google Scholar, 4.Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-233Crossref PubMed Scopus (773) Google Scholar, 5.Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Abstract Full Text PDF PubMed Google Scholar, 6.Mukoyama M. Nakajima M. Horiuchi M. Sasamura H. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Abstract Full Text PDF PubMed Google Scholar). Two closely related AT1 isoforms (AT1A and AT1B) have been identified in rat and mouse species(7.Iwai N. Inagami T. FEBS Lett. 1992; 298: 257-260Crossref PubMed Scopus (381) Google Scholar, 8.Sasamura H. Hein L. Krieger J.E. Pratt R.E. Kobilka B.K. Dzau V.J. Biochem. Biophys. Res. Commun. 1992; 185: 253-259Crossref PubMed Scopus (279) Google Scholar). AT1 receptors have been shown to be coupled to G-proteins and to activate phospholipase C (PLC)(9.Bottari S.P. de Gasparo M. Steckeling U.M. Levens N.R. Frontiers Neuroendocrinol. 1993; 14: 123-171Crossref PubMed Scopus (338) Google Scholar, 10.Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar). This results in inositol trisphosphate (IP3) generation, which then causes an increase in intracellular calcium concentrations, and diacylglycerol formation, which leads to protein kinase C activation(11.Farese R.V. Larson R.E. Davis J.S. Endocrinology. 1984; 114: 302-304Crossref PubMed Scopus (63) Google Scholar, 12.Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3297) Google Scholar).The molecular location of the ligand binding domain of the G-protein-coupled receptors has been intensively investigated using genetic, biochemical, and biophysical approaches. The binding site for small molecules such as the bioamine neurotransmitters involves polar residues of the transmembrane domains (TM)(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). In contrast, the binding site for large hormones such as the pituitary glycoproteins is located in the large amino-terminal extracellular domain of the corresponding receptors(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The ligand binding domains of peptide receptors, such as AT1, have been more recently investigated. Peptide binding by AT1 receptors is dependent on the presence of four extracellular cysteines (14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar) as well as on several additional residues located in the extracellular domains(15.Hjorth S.A. Schambye H.T. Greenlee W.J. Schwartz T.W. J. Biol. Chem. 1994; 269: 30953-30959Abstract Full Text PDF PubMed Google Scholar). Whereas the binding of non-peptidic ligands is unaffected by these extracellular mutations, numerous polar residues of the hydrophobic transmembrane segments are determinant in binding of non-peptidic antagonists(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 17.Ji H. Leung M. Zhang Y. Catt K.J. Sandberg K. J. Biol. Chem. 1994; 269: 16533-16536Abstract Full Text PDF PubMed Google Scholar, 18.Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar, 19.Schambye H.T. Hjorth S.A. Bergsma D.J. Sathe G. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7046-7050Crossref PubMed Scopus (116) Google Scholar). Two of these residues, Asp74 and Tyr292, also play an essential role in the coupling of AT1A to PLC(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 18.Marie J. Maigret B. Joseph M.P. Larguier R. Nouet S. Lombard C. Bonnafous J.C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar).Biochemical analysis (20.Desarnaud F. Marie J. Lombard C. Larguier R. Seyer R. Lorca T. Jard S. Bonnafous J.C. Biochem. J. 1993; 289: 289-297Crossref PubMed Scopus (27) Google Scholar) and molecular modeling studies (21.Underwood D.J. Strader C.D. Rivero R. Patchett A.A. Greenlee W. Prendergast K. Chem. Biol. 1994; 1: 211-221Abstract Full Text PDF PubMed Scopus (65) Google Scholar) 2P. Broto, unpublished results. indicate a major role for the third and fifth transmembrane segments (TM-III and TM-V) in AngII binding. To define the functional roles of polar residues in these transmembrane domains, of different polar residues Ala and were in the rat AT1A receptor The of the receptors for peptidic and non-peptidic and as well as signaling were of the mutants were for the binding of AngII and analogs. the were located in different domains of the AT1A investigated the recently of be for this peptide hormone This was for different mutants of the receptors as well of the and receptors they were that molecular was transmembrane domains of two different B.K. Kobilka K. R.J. PubMed Scopus Google Scholar, R. J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R. J. FEBS Lett. 1993; PubMed Scopus Google Scholar). The ligand binding and signaling of four binding AT1A mutants were in COS in different