TM2-TM7 Interaction in Coupling Movement of Transmembrane Helices to Activation of the Angiotensin II Type-1 Receptor

Agonist-induced rigid body motion of transmembrane (TM) helices has been established as a unifying mechanism in the activation of the G protein-coupled receptors. In attempts to measure specific conformational transitions during the activation of the type 1 receptor for angiotensin II (AT1), we found a decrease in accessibility of Cys76 in the second TM helix, suggesting that the orientation of TM2 is altered (Miura, S., and Karnik, S. S. (2002) J. Biol. Chem. 277, 24299–24305). Now we provide evidence that the TM2 helical movement plays a role in regulating the activated state of the AT1 receptor, and this role may involve an interaction between TM2 and TM7. Alanine substitution of native Cys296in TM7 leads to increased accessibility of Cys289 and diminished response to bound agonist. Both effects of the C296A mutation are suppressed when combined with F77A and N111G mutants. The TM7 conformation and the sensitivity of Cys289 altered by C296A mutation are suppressed by the F77A mutation in TM2 to salvage function. We show that the F77A mutant alters orientation of both TM2 and TM7 but does not induce constitutive activity in suppressing the C296A mutant effects. Thus, interaction of TM2 and TM7 is important for transmembrane signal transduction in the AT1 receptor. Agonist-induced rigid body motion of transmembrane (TM) helices has been established as a unifying mechanism in the activation of the G protein-coupled receptors. In attempts to measure specific conformational transitions during the activation of the type 1 receptor for angiotensin II (AT1), we found a decrease in accessibility of Cys76 in the second TM helix, suggesting that the orientation of TM2 is altered (Miura, S., and Karnik, S. S. (2002) J. Biol. Chem. 277, 24299–24305). Now we provide evidence that the TM2 helical movement plays a role in regulating the activated state of the AT1 receptor, and this role may involve an interaction between TM2 and TM7. Alanine substitution of native Cys296in TM7 leads to increased accessibility of Cys289 and diminished response to bound agonist. Both effects of the C296A mutation are suppressed when combined with F77A and N111G mutants. The TM7 conformation and the sensitivity of Cys289 altered by C296A mutation are suppressed by the F77A mutation in TM2 to salvage function. We show that the F77A mutant alters orientation of both TM2 and TM7 but does not induce constitutive activity in suppressing the C296A mutant effects. Thus, interaction of TM2 and TM7 is important for transmembrane signal transduction in the AT1 receptor. transmembrane angiotensin II [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-COO−] angiotensin II type-1 methanethiosulfonyl methanethiosulfonyl ethyl-ammonium methanethiosulfonyl trimethyl-ammonium methanethiosulfonylethyl-sulfonate inositol phosphate wild-type G protein-coupled receptor Recent advances in the human genome project has yielded an estimate of > 2000 transmembrane (TM)1 receptors that are members of the G protein-coupled receptor (GPCR) superfamily. Diverse endogenous ligands such as endocrine hormones, neurotransmitters, ions, peptides, proteases, glycoproteins, and sensory signals can activate these receptors (1Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (629) Google Scholar). Activated GPCRs recruit intracellular heterotrimeric G proteins and stimulate GTP/GDP exchange to initiate receptor-specific signals. Although, their structure-function relationships vary markedly, a seven TM helical structure is essential for signal transduction by GPCRs. Therefore, activation of GPCRs has been proposed to involve a common molecular mechanism i.e.rigid body movement of TM helices (2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google Scholar). Previous studies (1Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (629) Google Scholar, 2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google Scholar) of prototypical GPCRs demonstrated that activation induces relative movement of TM3, TM6, and TM7. The type 1 (AT1) receptor for the octapeptide hormone angiotensin II (Ang II) is a member of the GPCR superfamily (Fig.1). It is an important target for drug development because abnormalities in its function are linked to hypertension, water-electrolyte imbalance, hyperaldosteronism, cardiac hypertrophy, and heart failure (4De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger T. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). Activation of AT1receptor by Ang II and its subsequent coupling to G proteins, Gq/11, results in phospholipase C (PLC) activation, inositol phosphate accumulation, and smooth muscle contraction (4De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger T. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). Ang II binding involves TM3, TM5, TM6, and TM7 helices. Following binding, interaction of Ang II-Tyr4 with Asn111and Ang II-Phe8 with His256 (see Fig. 1) leads the AT1 receptor from an inactive (R) to activated state (R*) (5Karnik S.S. Husain A. Graham R.M. Drugs, Enzymes and Receptors of the Renin-Angiotensin System: Celebrating a Century of Discovery. Harwood Academic Press, Amsterdam, The Netherlands2000: 117-130Google Scholar, 6Hunyady L. Gaborik Z. Vauquelin G. Catt K.J. J. Ren. Ang. Aldo. System. 2001; 2: S16-S23Crossref PubMed Scopus (14) Google Scholar). Substitution of Asn111 in the TM3 results in constitutive activation of the receptor (5Karnik S.S. Husain A. Graham R.M. Drugs, Enzymes and Receptors of the Renin-Angiotensin System: Celebrating a Century of Discovery. Harwood Academic Press, Amsterdam, The Netherlands2000: 117-130Google Scholar). The conservation of agonist-receptor contacts and induction of constitutive activation indicates that Ang II may activate AT1 receptor by inducing rigid body motion of TM helices, as in the case of other GPCRs. However, the specific conformational changes essential for activation of the AT1 receptor are mostly unknown. Recently, we showed that reduction of Asn111 side chain size in the AT1 receptor resulted in different degrees of constitutive activation (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Reporter Cys-accessibility mapping (RCAM) in these mutants demonstrated different degrees of accessibility of the TM2 residue, Cys76, to methanethiosulfonyl-ethylaminoacetate (MTSEA) derivatization. This provided the first evidence of TM2 helical movement during AT1 receptor activation. An exhaustive mapping of conformational changes in other helices was, however, hampered because the MTSEA reactivity of the remaining native Cys residues is not known. Therefore, we reasoned that the construction of an AT1receptor CYS− mutant that lacks all free Cys residues would be a useful template for mapping the conformational changes in all TM helices. Hence, we constructed and evaluated single or combinatorial replacements of free Cys residues in the AT1receptor. We unexpectedly discovered that substitution of native Cys296 induces a change in the orientation of TM7, accompanied with functional defects. We show here that both effects induced by C296A mutation are abolished in a mutant located in the TM2. Taken together, these findings expose a critical role of TM2-TM7 interaction in coupling helical movements to G protein activation. The highly reactive, sulfhydryl-specific alkylating reagents used were CH3SO2-SCH2CH2NH3+(methanethiosulfonyl ethyl-ammonium [MTSEA+], adduct size about 4.726 Å), CH3SO2-SCH2CH2NMe3+(methanethiosulfonyl trimethylammonium [MTSET+], adduct size about 6.058 Å), CH3SO2-SCH2CH2SO3−(methanethiosulfonylethyl-sulfonate [MTSES−]), and CH3SO2-SCH (methanethiosulfonyl-methyl), which were purchased from Toronto Research Chemicals, Inc., Ontario, Canada. [Sar1,Ile8]Ang II and [Sar1]Ang II were purchased from Bachem (Torrance, CA).125I–[Sar1,Ile8]Ang II, (specific activity 2200 Ci/mmol) was purchased from Dr. Robert Speth, Washington State University (Pulman, WA). Losartan was a gift from DuPont Merck Co. (Wilmington, DE). The synthetic rat AT1 receptor gene, cloned in the shuttle expression vector pMT-2, was used for expression and mutagenesis as described in our earlier studies (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To express the AT1 receptor protein, 10 μg of purified plasmid DNA per 107 cells was used in transfection. COS1 cells (American Type Culture Collection, Rockville, MD), cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, were transfected by the DEAE-dextran method. Transfected cells cultured for 72 h were harvested, and cell membranes were prepared by the nitrogen Parr bomb disruption method in the presence of protease inhibitors. The final membrane suspension was at 1 mg/ml protein. The receptor expression was assessed in each case by immunoblot analysis (not shown) and by125I–[Sar1,Ile8]Ang II saturation-binding analysis. 125I–[Sar1,Ile8]Ang II-binding experiments were carried out under equilibrium conditions, as previously described (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Membranes expressing the wild-type (WT) or the mutant receptor were incubated with 300 pm125I–[Sar1,Ile8]Ang II for 1 h at 22 °C in a 250 μl volume. Nonspecific binding of the radioligand was measured in the presence of 1 mm127I–[Sar1,Ile8]Ang II. The binding experiments were stopped by filtering the binding mixture through Whatman GF/C glass fiber filters, which were extensively washed further with binding buffer. The bound ligand fraction was determined from the counts per min remaining on the membrane. Binding kinetics were determined using the computer program Ligand®. TheK d and B max values represent the mean ± S.E. of three to five independent determinations. Aliquots of cell membranes (20 μl) were incubated with or without MTS reagents at the stated concentrations (0.1–12.5 mm) at 22 °C for the indicated time (2–10 min) in 20 mm HEPES buffer (pH 7.4). The was with buffer to the and for 10 min at at in was used II binding analysis. The II binding was using the binding binding without The values represent the mean ± S.E. of to independent determinations. COS1 cells transfected in were for h with specific activity 22 at °C in Dulbecco's modified essential medium 10% fetal bovine The cells were washed three with and incubated with 10 for 20 were and for min at the of the medium was and the was from the cells by the as described previously (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The values represent the mean ± S.E. of three to independent determinations. of native Cys residues to ligand was measured by a highly reactive, sulfhydryl-specific with Cys residues and a with which is > in membrane (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, M. A. PubMed Scopus Google Scholar, S.T. Biochemistry. PubMed Scopus Google Scholar, G. L. U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This results in the of a Cys a the Cys is in the ligand the with II binding through or of the wild-type AT1 receptor to for min abolished specific binding of the II by The kinetics of is on the of the MTS and the time of on the in a with was used for the analysis of mutants. Binding by MTSEA is and leads to a decrease in the B max We earlier that the and the not II binding because these reagents with the wild-type AT1 receptor. In the presence II, the AT1 receptor was with (see S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To or native Cys of the AT1receptor that with MTS reagents to binding we Cys residues with residues located in the TM and and are to be not in CYS− mutant AT1receptor that all Cys residues was Cys mutants and three Cys mutants resulted during the construction of CYS− mutant AT1 receptor (see The of these mutants was not different from that of the receptor II that single or not the conformation of the receptor. We that of the proposed for the AT1 receptor in (4De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger T. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar) is and for a functional receptor and the other native Cys residues are not to be in ligand This indicates that native Cys residues in the AT1 receptor be used as conformational B max and and values for wild-type and mutant 1 of values represent inositol measured in COS1 cells transfected with and mutant AT1 receptor expression μg in three independent measured in COS1 cells transfected with expression plasmid without the AT1 receptor to activated was from all The of [Sar1]Ang II) values for the AT1 receptor was ± receptor. This was as to represent the of different on the function of AT1 receptor. The expression of receptor for different mutants ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± values are and for independent for each In all experiments an II was used to receptor in the The specific binding was to be 10% of to each and the binding was of specific The values represent inositol measured in COS1 cells transfected with and mutant AT1 receptor expression μg in three independent measured in COS1 cells transfected with expression plasmid without the AT1 receptor to activated was from all The of [Sar1]Ang II) values for the AT1 receptor was ± receptor. This was as to represent the of different on the function of AT1 receptor. The expression of receptor for different mutants in a The values are and for independent for each In all experiments an II was used to receptor in the The specific binding was to be 10% of to each and the binding was of specific with mm for min binding by ± in the and mutant receptors. In the and CYS− binding was to with and findings are with our earlier that II binding is to the of with Cys76 in the TM2 helix, and the remaining free Cys residues are not (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The mutant sensitivity to but the of its reactivity is In both the and of with the in the C296A mutant receptor, suggesting that this mutation may the conformation of the receptor and binding studies with the AT1 an reduction of for C296A with the receptor. Substitution of Cys296 with and the for mutants coupling to Previous mutagenesis L. Gaborik Z. Vauquelin G. Catt K.J. J. Ren. Ang. Aldo. System. 2001; 2: S16-S23Crossref PubMed Scopus (14) Google Scholar) studies indicated that TM7 plays an important role in and signal activation. on these we that C296A mutation receptor to increased accessibility of or native Cys The accessibility of remaining native Cys residues in with the C296A mutation was in Fig. the C296A mutant at The binding II is not to MTSEA when both Cys76 and Cys289 are in the C296A of of binding was in and mutants with the C296A The reduction is with of the Cys76 residue, which is The of the mutant with that of the mutants and C296A indicates that the remaining is with reactivity of Cys289 in these mutants The analysis indicates that the C296A mutation induced an in the accessibility of Cys289 without the accessibility of The of C296A mutation was in a of the AT1 receptor and S. Karnik, constructed on of bovine (1Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (629) Google Scholar, K. T. T. Behnke C.A. D.C. Okada T. Stenkamp R.E. M. M. 2000; PubMed Scopus Google Scholar). In our the Cys296 side chain is located in an of the that and its side chain is with indicated to the TM7 that would interaction with the The C296A mutation an increased sensitivity of Cys289 through change of without other native Cys residues in the AT1 receptor. We that the of Cys289 to C296A mutation a conformational change of TM7 in movement of Cys289 the evidence for a specific interaction between TM2 and TM7 in the AT1 receptor was in an earlier J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with this the AT1 receptor molecular indicated that the TM7 the Cys296 extensively with TM2. Therefore, we for in the TM2 and found that and of in TM2 the of Cys289 (see Fig. a decrease of MTSEA of Cys76 is not to MTSEA in these mutants as is in the This is by the of the mutants and We that side for the of Cys76, a that is In the AT1 receptor molecular the side chain of is located at the between the ligand and protein with to residues located on TM7. Therefore, residues for a of both TM2 and TM7 the TM to the in the protein the the conformational Cys76 on TM2 be in not of Cys76 in the F77A mutants is with the the residues for TM7 conformation is not The MTSEA in the F77A mutant does not a conformational change in TM7. the Cys289 in TM7 is not (see Fig. 1 a further decrease in the in the is to An in its reactivity be but not found in these mutants. The mutant MTSEA which is with that of the receptor. The of Cys289 induced by the C296A mutation and the accessibility of Cys76 by the F77A mutation are in this the MTSEA of the mutant demonstrated that of II binding is to with The of the mutant indicated that of is to accessibility of the change in was accompanied by an of for and of in to the C296A mutant of other native Cys residues not change Thus, the of mutant are or The effects by F77A and C296A on TM2 and TM7 out in the The conformation of the TM helical in this mutant the of helices in the receptor as by of Cys76 and the sensitivity of In interaction between TM3 and was from effects of and was by the structure (2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google Scholar). an between of TM3 and was demonstrated in the receptor (2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, G. L. U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). interaction between TM2 and TM7 is in GPCRs. and between TM2 and TM7 been in the structure of bovine (1Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (629) Google Scholar, K. T. T. Behnke C.A. D.C. Okada T. Stenkamp R.E. M. M. 2000; PubMed Scopus Google Scholar). The reactivity of Cys76 is a of activated receptor conformation in AT1 receptor (7Miura S. Karnik S.S. J. Biol. Chem. 2002; 277: 24299-24305Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Hence, the decrease of altered function. To the functional of the motion of helices in and we measured of in COS1 cells expressing the and receptors. To for of relationships to receptor per receptor was in each case (see The [Sar1]Ang II ± was not different between the and with these the F77A mutant a of ± 1 and a mutant with was S. S. U. S. A. 2000; PubMed Scopus Google Scholar). To this change is to constitutive activation, the were measured at different receptor The of to for the ± was not different from that for the F77A mutant ± Thus, the F77A mutation increased the ligand but does not to induce constitutive In in the C296A mutant both the of [Sar1]Ang II ± and response are that the C296A is to receptor activation. This that the TM7 motion and of Cys289 is a for receptor activation. We evaluated these effects on the constitutive activity of the N111G receptor. activation results from of a of for conformational changes essential for receptor activation (5Karnik S.S. Husain A. Graham R.M. Drugs, Enzymes and Receptors of the Renin-Angiotensin System: Celebrating a Century of Discovery. Harwood Academic Press, Amsterdam, The Netherlands2000: 117-130Google Scholar). of intracellular in the COS1 as earlier from this The of this activity was ± not by of F77A ± and ± The constitutive activity of N111G receptor was not abolished by of C296A reduction of the activity was in the mutant ± The [Sar1]Ang response was in the mutant but was not different in the remaining three mutants (see Fig. The F77A conformation of the receptor with the as increased of [Sar1]Ang II. However, the of on the constitutive activity of N111G mutant indicates that does not in the of receptor activation. the F77A mutation the activation, for the response in both and the N111G We that response of the receptor, and this role may on the of TM2 to with TM7. The here that TM2 and TM7 are a of functional helical motion in function. a decrease of indicates TM2 motion in of activated receptor an of indicates receptor of our is to conformational for each and in the AT1 receptor to of functional of the receptor in a cell free here that a mechanism for TM2-TM7 interaction is in coupling binding to receptor activation. between in TM2 and in TM7 in the AT1 receptor was the of this interaction was proposed that TM2 and TM7 helices to each other in the activated state J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The interaction proposed in the earlier was not found in our of the AT1 receptor. The out a interaction between and the residues in this However, the structure in our the for interaction between these helices. The TM2-TM7 in the involves residues and with side an between and and between TM2 and TM7. Previous mutagenesis studies and that these residues activation or and the for ligands L. Gaborik Z. Vauquelin G. Catt K.J. J. Ren. Ang. Aldo. System. 2001; 2: S16-S23Crossref PubMed Scopus (14) Google Scholar, J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. S. U. S. A. 2000; PubMed Scopus Google Scholar, U. S. A. PubMed Scopus Google Scholar). studies the of TM7 as a for the interaction of the side chain of Ang II J. M. M. L. G. Biochemistry. 2002; Scopus Google Scholar). Therefore, we that about by to the and are to induce motion of these helices. It to that relative movement of both TM2 and TM7 is important for of receptor function. a of GPCRs are of signal transduction in response to been in The mechanism by which the binding of ligand leads to activation of the receptor is to be (2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google Scholar). studies carried out in prototypical receptor, receptor, and receptors that receptor activation involves critical conformational changes in TM3 and (2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google Scholar, 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google Scholar). In both and receptor, studies provided evidence for a rigid body motion of the TM helices. motion G protein activation, that movement of these TM helices is critical for activation. activation binding of the receptor activation can be in GPCRs. This of activation involves movement of TM3 and TM6, suggesting that movements may be members of GPCR movements in the to and in TM7, been in an between TM3 and TM7, motion of these helices is in the receptor. changes been for and The established motion of TM3 and in three however, does not that movement of other may not to receptor activation. In studies of the receptor, evidence for conformational changes was U. S. A. 2001; PubMed Scopus Google which is of in the of conformational changes in the activation of GPCRs may involve and specific movements of TM helices to with time (see 2Gether U. Endo. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1113) Google and 3Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (281) Google for the that residues to in TM2 and the in TM7 of the AT1 receptor are highly in the GPCR the functional of TM2-TM7 in this may be common to GPCRs. of TM2-TM7 has been in the receptor, hormone receptor, receptor, receptor, and receptor J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the effects we here on of receptor and of state is a common in GPCRs further We and for in

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