Dependence of AT1 Angiotensin Receptor Function on Adjacent Asparagine Residues in the Seventh Transmembrane Helix

  1. László Hunyady1,
  2. Hong Ji2,
  3. Gowraganahalli Jagadeesh3,
  4. Meng Zhang2,
  5. Zsuzsanna Gáborik1,
  6. Balázs Mihalik1 and
  7. Kevin J. Catt2
  1. 1Department of Physiology, Semmelweis University of Medicine, H-1088 Budapest, Hungary (L.H., Z.G., B.M.), 2Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510 (H.J., M.Z., K.J.C.), and 3Division of Cardio-Renal Drug Products, Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20857 (G.J.)
     
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    Abstract

    For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1,Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.

    The AT1 angiotensin receptor is a GPCR that mediates the physiological actions of the octapeptide pressor hormone Ang II (Spät et al., 1991; Ganguly and Davis, 1994;Griendling et al., 1996). GPCRs share a common basic structure of seven transmembrane helices connected by alternating intracellular and extracellular loops. All members of this receptor family couple to specific G proteins that mediate the activation of several plasma membrane effector systems (Probst et al.,1992; Strader et al., 1994). AT1receptors, which occur as highly homologous and functionally similar AT1a and AT1b subtypes in rodents, are primarily coupled through the Gq/11group of G proteins to stimulation of phospholipase C activity and Ins(1, 4, 5)P3-induced Ca2+signal generation. This is usually accompanied by activation of protein kinase C, mediated by increased diacylglycerol production in concert with the agonist-evoked intracellular Ca2+ signal (Spät et al., 1991;Ganguly and Davis, 1994; Griendling et al., 1996). The molecular mechanisms by which activated GPCRs are coupled to signal generation are currently the focus of intensive studies investigating their structure-function relationships.

    In many peptide hormone receptors, the cognate physiological ligands bind to a site formed by the extracellular loops and the outermost regions of the transmembrane helices (Strader et al.,1995; Hunyady et al., 1996). The conformational changes evoked by agonist binding are transmitted by the transmembrane helices to the intracellular loops, which are believed to comprise the regions that couple the receptor to its intracellular signaling systems. The current structural information about GPCRs is based on low-resolution maps of bovine and frog rhodopsins (Schertler et al., 1993;Schertler and Hargrave, 1995). These studies, together with recent data obtained by electron cryomicroscopy of crystals of frog rhodopsin (Unger et al., 1997), have confirmed the seven-transmembrane helix nature of GPCRs and have provided information about the positioning of the helices but have not elucidated the structural changes that are involved in the mechanism of activation of these receptors. For this reason, several theoretical models of the three-dimensional conformation of the AT1receptor and other GPCRs have been constructed (Baldwin, 1993; Donnellyet al., 1994; Joseph et al., 1995; Underwoodet al., 1995; Yamano et al., 1995; Inoue et al., 1997). Although predictions based on such studies have been helpful in defining the binding sites of several GPCRs, the manner in which agonist binding changes the conformation of the receptor molecule has not been clarified.

    Pharmacological studies have identified two major classes of nonpeptide compounds that bind selectively to angiotensin AT1 and AT2 receptors (Griendling et al., 1996). Structure-function studies have shown that the binding site of such nonpeptide antagonists is located within the intramembrane binding pocket, with contributions from several residues located in the transmembrane helices (Ji et al., 1994; Hunyady et al., 1996; Inoue et al., 1997; Karnik et al., 1997). More recent studies on nonpeptide analogs have led to the identification of compounds that act as partial agonists of the AT1 receptor, causing increased blood pressure in vivo and stimulation of signal transduction in vitro (Perlman et al., 1995;Kivlighn et al., 1996). Mutational studies focusing primarily on the extracellular regions have failed to identify residues that contribute to the binding of nonpeptide agonists (Perlman et al., 1995). However, the nonpeptide agonists and antagonists are closely related chemically, and it is likely that some of the residues that participate in the binding of nonpeptide antagonists also contribute to the binding of nonpeptide agonists.

    The seventh transmembrane helix has been suggested to be an important area for both ligand binding and activation of the AT1 receptor and several other GPCRs (Luoet al., 1994; Marie et al., 1994; Hunyadyet al., 1995, 1996; Strader et al., 1995; Laporteet al., 1996; Inoue et al., 1997). Mutations of amino acid residues in the seventh helix have been reported to interfere with the binding of nonpeptide Ang II antagonists. These residues include Tyr292 (the amino acid located in the position at which the retinal chromophore binds covalently to the rhodopsin molecule), Leu300 and Phe301 (two apolar amino acids located in the center of the conserved NPXXY sequence), and Asn294 and Asn295 (two polar residues located between Tyr292 and the NPXXY sequence) (Marieet al., 1994; Schambye et al., 1994b; Ji et al., 1995; Hunyady et al., 1996; Inoue et al., 1997; Karnik et al., 1997). In addition, mutations in the seventh transmembrane helix have been found to interfere with the binding of peptide ligands and with signal transduction from the receptor (Marie et al., 1994; Hunyady et al.,1995; Ji et al., 1995; Laporte et al., 1996).

    Recent studies have suggested that an interaction between Asn295 and Asn111 in the third helix stabilizes the inactive conformation of the receptor, based on the finding that mutation of Asn295 or Asn111 caused constitutive activation of the AT1 receptor (Nodaet al., 1996; Balmforth et al., 1997; Groblewskiet al., 1997). The Asn111 residue has also been reported to interact with the side chain of Tyr4 of Ang II, and this has been suggested to initiate the process of receptor activation (Noda et al., 1996). Because the available structure-function data suggest that the seventh transmembrane helix of the AT1angiotensin receptor is a region in which the binding sites of peptide and nonpeptide ligands overlap (Hunyady et al., 1996), the role of this region in the binding of nonpeptide agonists was analyzed. Receptors bearing single amino acid substitutions of two polar residues (Asn294 and Asn295) and an adjacent apolar residue (Phe293) were created and expressed in COS-7 cells to study the role of this region in the ligand binding and activation of the AT1receptor.

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    Materials and Methods

    Mutagenesis of rat smooth muscle AT1a receptor cDNA.

    The rat AT1a receptor cDNA was subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen, San Diego, CA), as described earlier (Hunyady et al., 1994). The AT1a receptors used in this study also contained an octapeptide tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) inserted after the starting methionine of mutant and wild-type rat AT1a receptors, to permit the immunodetection of binding-deficient mutant receptors. In accordance with a recent report (Hein et al., 1997), the octapeptide tag had no major effect on the binding and inositol phosphate signaling properties of the wild-type rat AT1a receptor. The amino acid numbers used in this study refer to the positions in the published rat AT1a receptor sequence (Murphy et al.,1991); the presence of the octapeptide tag does not affect the numbering. Mutant rat AT1a receptors were created using the Mutagene kit (Bio-Rad, Hercules, CA). Each mutant contained a silent restriction site to facilitate the screening of colonies. Oligonucletides were from Midland Certified Reagent Co. (Midland, TX). All mutations were verified by dideoxy sequencing using Sequenase II (Amersham-USB, Arlington Heights, IL).

    Transient expression of AT1a receptor mutants.

    COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium containing 2 mml-glutamine, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. For measurements of inositol phosphate responses or [Sar1,Ile8]Ang II binding to intact cells, the cells were seeded in 24-well plates (50,000 cells/well) 3 days before transfection. Transient transfection was performed by replacing the culture medium with 0.4-ml aliquots of Opti-MEM I containing 8 μg of Lipofectamine and 1 μg of plasmid DNA for each sample. The cells were incubated for 5–6 hr in this solution, which was then replaced with culture medium. All experiments were performed 48 hr after the initiation of the transfection procedure.

    [Sar1,Ile8]Ang II binding to intact cells.

    To determine the surface expression level and structural integrity of the mutant receptors, the number of Ang II binding sites was determined by incubating the transfected cells with125I-[Sar1,Ile8]Ang II (0.05–0.1 μCi/sample) and increasing concentrations of unlabeled [Sar1,Ile8]Ang II in medium 199 (with HEPES) for 6 hr at 4°. The cells were washed twice with ice-cold Dulbecco’s phosphate-buffered saline, and the radioactivity associated with the cells was measured by γ-counting after solubilization with 0.5 m NaOH/0.05% SDS. The displacement curves were analyzed with the Ligand computer program, using a one-site model, as described earlier (Hunyady et al., 1995).

    Binding to COS-7 cell membranes.

    COS-7 cells were seeded in 15-cm tissue culture dishes 72 hr before the transfection, which was performed by the calcium phosphate precipitation method (Life Technologies, Gaithersburg, MD), using 50 μg of DNA. After 48 hr, the cells were washed, scraped into 1.5 ml of ice-cold 10 mmTris·HCl, pH 7.4, 1 mm EDTA, and then lysed by freezing. Crude membranes were prepared by centrifuging the samples at 16,000 × g. The pellet was resuspended in binding buffer (containing 100 mm NaCl, 5 mmMgCl2, and 20 mm Tris·HCl, pH 7.4), and the protein content was determined. Binding assays were performed at 25° in 0.2 ml of binding buffer supplemented with 2 g/liter BSA, except when nonpeptide agonists were tested. In these experiments, BSA was replaced with 0.1% lysozyme. Saturation binding experiments with125I-Ang II and125I-[Sar1,Ile8]Ang II were performed by adding increasing concentrations of the respective radioligand (up to 2 nm) to 15–30 μg of crude membranes. In displacement studies, each sample contained 0.05–0.1 μCi of125I-[Sar1,Ile8]Ang II, 15–30 μg of crude membranes, and the indicated concentrations of unlabeled ligand. G protein coupling was evaluated by measuring the binding of 125I-Ang II (0.05–0.1 μCi) under similar conditions in the presence of the indicated concentrations of GTPγS. After 90-min incubations at 25°, the unbound tracer was removed by rapid filtration, and the bound radioactivity was measured by γ-counting.

    Inositol phosphate measurements.

    In these experiments, the culture medium was replaced, 24 hr after transfection, with 0.5 ml of inositol-free Dulbecco’s modified Eagle’s medium containing 1 g/liter BSA, 20 μCi/ml [3H]inositol, 2.5% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin, as described earlier (Hunyady et al., 1994). After a 24-hr labeling period, the cells were washed twice and incubated in inositol-free modified medium 199 (with HEPES), in the presence of 10 mm LiCl, for 30 min at 37° and were then stimulated with the indicated concentration of Ang II for 20 min. Incubations were stopped by the addition of perchloric acid (5%, v/v, final), and the inositol phosphates were extracted as described earlier (Hunyadyet al., 1995). After neutralization, the samples were applied to a Bio-Rad AG1X8 column. The columns were washed four times with 3 ml of water and twice with 3 ml of 0.2 m ammonium formate in 0.1 m formic acid, to remove inositol and inositol monophosphates. After these washing steps, the combined InsP2/InsP3 fractions were eluted with two 3-ml aliquots of 1 m ammonium formate in 0.1 m formic acid, and radioactivities were determined by liquid scintillation counting.

    Materials.

    The cDNA clone (pCa18b) of the rat smooth muscle AT1a receptor (Murphy et al., 1991) subcloned into the mammalian expression vector pCDM8 (Invitrogen) was kindly provided by Dr. Kenneth E. Bernstein (Department of Pathology, Emory University, Atlanta, GA). Restriction enzymes were obtained from Boehringer Mannheim (Indianapolis, IN) or New England Biolabs (Beverly, MA). Culture media were from Biofluids (Rockville, MD). The medium 199 used in these experiments was modified to contain 3.6 mm K+, 1.2 mmCa2+, 1 g/liter BSA, and 20 mm HEPES. Lipofectamine and Opti-MEM I were from Life Technologies. Losartan was a gift from Dr. P. C. Wong (DuPont, Wilmington, DE).L-162,313 and L-163,491 were kindly provided by Dr. W. J. Greenlee (Merck Research Laboratories, Rahway, NJ).125I-Ang II and125I-[Sar1,Ile8]Ang II were obtained from Hazleton Laboratories (Vienna, VA) or DuPont New England Nuclear Research (Boston, MA), and [3H]inositol was from Amersham (Arlington Heights, IL).

    Statistical analysis.

    The significance of changes in the binding affinities and inositol phosphate responses of the mutant AT1 receptors were determined by analysis of variance combined with Scheffe’s range test.

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    Results

    Binding properties of mutant and wild-type AT1areceptors.

    The wild-type and F293A, N294A, and N295A mutant AT1a receptors were expressed in COS-7 cells, to study their radioligand binding and signaling characteristics. The positions of the mutated amino acids in the seventh transmembrane helix of the rat AT1a receptor are shown in Fig.1. Binding affinities were determined by measuring the inhibition of binding of the radioiodinated peptide antagonist125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of the unlabeled nonpeptide antagonist (losartan), the peptide antagonist (Fig.2), or two nonpeptide agonists (L-162,313 and L-163,491) (Fig.3). TheKd values are shown in Table1. The binding affinities of the F293A receptor for [Sar1,Ile8]Ang II and losartan were not significantly different from those of the wild-type receptor. The N294A receptor had normal affinity for [Sar1,Ile8]Ang II, indicating that the structure of this mutant receptor was intact. However, its affinity for losartan was markedly reduced, with a 62-fold increase in Kd. The N295A mutant receptor exhibited diminished binding affinity for both [Sar1,Ile8]Ang II and losartan, with Kd values 16 ± 2 and 104 ± 6 times those of the wild-type receptor, respectively.

    Figure 1
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    Figure 1

    Two-dimensional representation of the AT1a receptor. The locations of the intramembrane helices are based on the model of Baldwin (1993). The most conserved residues in the GPCRs are shown (white letters on black background). The amino acid residues analyzed in this article and the putative glycosylation sites of the receptor are also shown.

    Figure 2
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    Figure 2

    Inhibition of125I-[Sar1,Ile8]Ang II binding by [Sar1,Ile8]Ang II (upper) or losartan (lower). The binding of125I-[Sar1,Ile8]Ang II to the membranes of COS-7 cells expressing wild-type (•) or F293A (■), N294A (▵), or N295A (○) mutant AT1a receptors was measured in the presence or absence of the respective antagonists. All values are expressed as percentages of theB0 values determined in the absence of unlabeled ligand. Data are shown as means of duplicate determinations from a representative example of three to five experiments with similar results. Kd values are shown in Table 1.

    Figure 3
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    Figure 3

    Inhibition of125I-[Sar1,Ile8]Ang II binding by the nonpeptide agonist L-162,313 (upper) orL-163,491 (lower). The binding of125I-[Sar1,Ile8]Ang II to the membranes of wild-type (•) or F293A (■), N294A (▵), or N295A (○) mutant AT1a receptor-transfected COS-7 cells was measured in the presence or absence of the respective nonpeptide agonists. All values are expressed as percentages of theB0 values measured in the absence of unlabeled ligand. Data are shown as means of duplicate determinations from a typical experiment representing four experiments with similar results. Kd values are shown in Table 1.

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    Table 1

    Binding affinities of wild-type and mutant AT1a receptors for antagonist and nonpeptide agonist ligands

    Mutations of the adjacent asparagine residues in the seventh transmembrane helix also affected the binding affinities for the two nonpeptide agonists (Fig. 3, Table 1). The F293A mutation caused no significant change in the binding of these compounds. TheKd values of the N294A receptor forL-162,313 and L-163,491 were 3.9- and 2.8-fold higher, respectively, than those of the wild-type receptor. However, the affinities of the N295A mutant receptor for these nonpeptide agonists were more severely impaired, with Kd values 26- and 25-fold higher, respectively, than those of the wild-type receptor.

    The structural requirements at position 295 for ligand binding to the AT1a receptor were further analyzed by measurement of the binding affinities of a series of substitution mutants for [Sar1,Ile8]Ang II, losartan, and the AT1-specific nonpeptide agonistL-163,491 (Table 1). The binding affinity for [Sar1,Ile8]Ang II was most severely impaired with the above-described alanine-substituted (N295A) mutant receptor. Substitution with amino acids with larger side chains had less effect on [Sar1,Ile8]Ang II binding to the receptor, regardless of the nature of the side chain introduced (Table 1). For example, the inhibitory effects of three apolar substitutions for Asn295 (N295A, N295V, and N295L) decreased with increasing side-chain length. Substitution with leucine, which has the largest apolar side chain, caused the least impairment (2.4-fold) of [Sar1,Ile8]Ang II binding, similar to the effect of the conserved substitution N295Q (2.0-fold). Replacement by valine, with its smaller side chain, further impaired [Sar1,Ile8]Ang II binding affinity (6.5-fold), similar to the effects of the N295S (6.3-fold) and N295T (5.9-fold) mutations, but to a lesser extent than replacement with alanine (16-fold). Substitution of Asn295 with a negatively charged residue (N295D) did not significantly impair receptor affinity for [Sar1,Ile8]Ang II, and substitution with lysine caused a relatively minor (3.7-fold) decrease in binding affinity.

    The structural requirements for the nonpeptide compounds losartan andL-163,491 were different from those for the peptide antagonist at position 295 of the AT1a receptor. Losartan binding affinity was reduced >3-fold by substitution with all tested amino acids. The most pronounced impairments were observed after substitution with alanine (70-fold), serine (72-fold), or glutamine (74-fold). Losartan binding was much less affected by substitution with threonine (4.5-fold) or valine (3.6-fold). Alanine substitution had the largest inhibitory effect on the binding of nonpeptide ligands, similar to the results obtained with [Sar1,Ile8]Ang II. The structural requirements for receptor binding of the nonpeptide agonistL-163,491 (Kaye et al., 1995) differed from those for losartan in several respects; the conserved substitution with glutamine in the N295Q receptor did not affect agonist binding, in contrast to its major effect on losartan binding. The binding ofL-163,491 was substantially reduced by substitution with serine (9.2-fold reduction) and was less affected by replacement with threonine, aspartic acid, lysine, leucine, or valine (2–4-fold) (Table1).

    Saturation binding studies.

    The affinities of the alanine mutant AT1a receptors for the physiological agonist Ang II and the peptide antagonist [Sar1,Ile8]Ang II were also evaluated in saturation binding experiments with125I-labeled radioligands (Table2). Under these conditions, Scatchard analysis of 125I-Ang II binding to the wild-type receptor detected only the high-affinity agonist binding site, with aKd of 0.12 nm. For this reason, the number of Ang II binding sites measured in COS-7 cells expressing the wild-type receptor is approximately 16% of the sites calculated from binding analysis with [Sar1,Ile8]Ang II, which binds with a single affinity (Kd = 0.23 nm) to the entire AT1receptor population (Table 2).

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    Table 2

    Binding parameters for wild-type and mutant AT1a receptors with125I-Ang II and 125I-[Sar1,Ile8]Ang II

    In accordance with the findings of the binding-inhibition studies, the F293A mutation had no major effect on Ang II binding, and the N295A mutation decreased the binding affinities for the two ligands to similar extents (7.5-fold). The affinity of the N294A receptor for Ang II showed a moderate decrease (3.2-fold), whereas its affinity for [Sar1,Ile8]Ang II was similar to that of the wild-type receptor. It is interesting to note that the reduction in the binding affinity of the N294A mutant AT1a receptor for Ang II was similar to the above-described impairment of its affinity for nonpeptide agonists.

    Inositol phosphate responses and G protein coupling of alanine mutant AT1a receptors.

    The signal transduction properties of the alanine mutant receptors were studied by measuring the inositol phosphate (InsP2 plus InsP3) responses of the expressed mutant receptors in [3H]inositol-prelabeled COS-7 cells in the presence of lithium. The data were normalized to the measured receptor levels, because under these conditions the combined InsP2 plus InsP3 response is directly proportional to the receptor number (Hunyady et al., 1995). Representative curves for the inositol phosphate responses of alanine mutant AT1a receptors are shown in Fig. 4. The amplitudes of the inositol phosphate responses for the F293A, N294A, and N295A mutant receptors were 107 ± 27%, 16.3 ± 0.7% (p < 0.01), and 73.0 ± 11.3%, respectively, compared with those of the wild-type AT1a receptor (three experiments). The EC50 values for the wild-type and F293A, N294A, and N295A mutant AT1a receptors for inositol phosphate responses were 0.91 ± 0.36 nm, 1.1 ± 0.3 nm, 7.3 ± 1.6 nm(p < 0.01), and 5.0 ± 1.1 nm(p < 0.05), respectively (three experiments). These data demonstrate that the reduced efficacy of the N294A mutant receptor for inositol phosphate signal generation was accompanied by decreased potency of Ang II to activate this receptor. The capacity of the F293A mutant receptor for inositol phosphate generation was similar to that of the wild-type receptor, whereas the decreased potency of the N295A mutant receptor is in accordance with its reduced affinity for Ang II. The maximal inositol phosphate response of the N295A mutant receptor did not differ significantly from that of the wild-type receptor. Thus, although the affinity of this mutant receptor was reduced, the peptide agonist could induce receptor activation when present at sufficiently high concentrations.

    Figure 4
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    Figure 4

    Inositol phosphate dose-response curves for AT1a receptors. The combined accumulation of radioactivity in the InsP2 plus InsP3 fraction of wild-type (•) or F293A (■), N294A (▵), or N295A (○) mutant AT1a receptor-transfected COS-7 cells was measured after [3H]inositol prelabeling, as described in Materials and Methods. Data were normalized to the number of expressed extracellular binding sites for each mutant, and stimulation is expressed as a percentage of the response mediated by the wild-type receptor with 1 μm Ang II (AII). Values are shown as means and ranges of duplicates from one of three similar experiments. In these experiments, basal versus stimulated (1 μm Ang II) values for InsP2 plus InsP3 labeling for wild-type, F293A, N294A, and N295A AT1a receptors were 932 ± 72 versus 8127 ± 1236, 935 ± 84 versus 7386 ± 1196, 1081 ± 21 versus 932 ± 117, and 804 ± 88 versus 7551 ± 2414 cpm, respectively (three experiments).

    The G protein coupling of the N294A receptor was also investigated, by measuring the effect of GTPγS on the binding of the physiological agonist Ang II. As reported earlier (Hunyady et al., 1994), GTPγS substantially reduced the binding of125I-Ang II to the wild-type AT1a receptor in membranes of transiently transfected COS-7 cells (Fig. 5). However, the inhibitory effect of GTPγS on Ang II binding was significantly decreased with the N294A mutant AT1a receptor (four experiments) (p < 0.001), in accordance with its impaired coupling to inositol phosphate generation.

    Figure 5
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    Figure 5

    Effects of GTPγS on the 125I-Ang II binding of wild-type (•) and N294A (▵) AT1a receptors. Membranes were prepared from COS-7 cells transfected with wild-type or N294A AT1a receptors, and the binding of125I-Ang II was measured in the presence or absence of the indicated concentrations of GTPγS, as described in Materials and Methods. Data are expressed as a percentage of the binding measured in the absence of GTPγS and are shown as means ± standard errors of values obtained in four independent experiments, each performed in duplicate. Errors not shown are within the data symbols. The reduced effect of GTPγS on agonist binding to the N294A mutant receptor was significant at the p < 0.01 level.

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    Discussion

    Alanine substitution of Asn294 affects agonist binding and signal transduction.

    In this study, mutational analysis has defined the functional properties of three adjacent amino acids located in the seventh transmembrane helix of the AT1 receptor. According to predictions based on modeling studies of GPCRs, the two consecutive polar residues (Asn294 and Asn295) are likely to face the interior of the AT1 angiotensin receptor, whereas Phe293 is expected to face the lipid environment of the plasma membrane (Baldwin, 1993; Donnelly et al., 1994). In accordance with these proposals, we found that alanine replacement of Phe293 had no major effect on angiotensin receptor function, whereas similar mutations of Asn294 and Asn295 significantly affected the binding and signaling functions of the receptor.

    The N294A receptor exhibited markedly reduced binding affinity for losartan and decreased affinities for peptide and nonpeptide agonists, but its affinity for [Sar1,Ile8]Ang II was completely normal. These data differ from those of earlier reports, in which mutations of several extracellular residues and alanine replacement of Asn294 did not affect the binding affinities of the human AT1 receptor for Ang II and two nonpeptide agonists, L-162,313 and L-162,782 (Perlmanet al., 1995,1997). The most prominent feature of the N294A receptor was its markedly attenuated capacity to engender inositol phosphate responses, even in the presence of high concentrations of Ang II. These data suggest that Asn294 is an important determinant of agonist-induced receptor activation and signal transduction. The moderately impaired agonist binding of the N294A receptor may reflect its inability to achieve the optimal conformation required for agonist-induced activation (Karnik et al.,1997). This conclusion is supported by the finding that the effect of GTPγS on Ang II binding was reduced with the N294A mutant AT1a receptor.

    Replacements of Asn295 affect ligand binding of the AT1a receptor without interfering with its activation.

    Alanine replacement of Asn295 caused a substantial decrease in receptor binding affinity for all AT1 receptor ligands tested, without significant impairment of the maximal inositol phosphate response to Ang II stimulation. The role of Asn295 in the binding function of the AT1a receptor was evaluated with a series of mutant AT1a receptors bearing other replacements of this residue. In general, substitution with amino acids with polar, apolar, or charged side chains had less effect on the binding affinities for the tested ligands than did alanine substitution. In the case of [Sar1,Ile8]Ang II, substitution with amino acids with smaller side chains (e.g., serine and threonine) caused more marked impairment of binding than did substitution with residues with larger side chains (e.g., aspartic acid, glutamine, leucine, or lysine). Among the substituents with apolar side chains, leucine, with the largest side chain, caused the least impairment of [Sar1,Ile8]Ang II binding. Conversely, substitution with alanine, which has the smallest side chain, caused the most marked reduction of binding affinity. There was no clear requirement regarding the nature of the amino acid substitution in position 295. Substitution with polar (glutamine), charged (aspartic acid or lysine), or apolar (leucine) residues caused moderate impairment of binding. These data suggest that the Asn295 residue has a structural effect on [Sar1,Ile8]Ang II binding, and they argue against a direct contact between the bound ligand and this residue.

    Losartan binding to the AT1a receptor was reduced by all tested substitutions of the Asn295 residue. These findings are in accordance with previous studies on the affinity of the N295S mutant AT1 receptor for losartan (Schambye et al., 1994a,b; Balmforth et al., 1997). The unique requirement for asparagine in position 295 for normal losartan binding may reflect a direct interaction between this ligand and Asn295 of the receptor. The intramembrane location of Asn295 and the effects of its replacement on the binding of peptide and nonpeptide antagonists suggest that this residue is essential for the integrity of the intramembrane binding pocket of the AT1 receptor.

    Another interesting feature of the N295A mutation was its deleterious effect on the binding of the nonpeptide agonists L-162,313 and L-163,491. This finding differs from those of previous studies, in which replacement of Asn295 by aspartic acid had no effect on Ang II and nonpeptide agonist binding (Perlman et al.,1995,1997). Although aspartic acid was found to substitute for Asn295 without detectable loss of binding affinity forL-162,313, the efficacy with which L-162,313 and other nonpeptide agonists stimulated inositol phosphate responses was greatly reduced with the N295D mutant (Perlman et al.,1997). In our study, the affinity of the N295D mutant receptor for the AT1-selective nonpeptide agonistL-163,491 was moderately impaired. All tested substitutions of Asn295 caused less impairment of L-163,491 binding, compared with that of losartan. In contrast to its marked effect on losartan binding, the conserved substitution of glutamine for Asn295 had no effect on the binding affinity for L-163,491. These data suggest that the structural requirements in position 295 for the binding of L-163,491 differ from those for losartan. Although these data do not provide evidence for a role of Asn295 in nonpeptide agonist binding, the marked inhibition of binding to the N295A and N295S mutant receptors suggests that the intact intramembrane binding pocket is an important requirement for the binding of nonpeptide agonists.

    Our data also showed that alanine substitution of Asn295 reduced the high-affinity binding of Ang II to the receptor. In accordance with this finding, the potency with which Ang II stimulated inositol phosphate responses in cells expressing the N295A receptor was also reduced. Although earlier studies suggested that mutation of Asn295 to serine causes a relatively minor change in the Ang II binding affinity of the receptor (Schambye et al., 1994a,b; Balmforthet al., 1997), the present data, in accordance with previous studies on the rat AT1b receptor (Ji et al., 1995), demonstrate that the N295A mutation also affects the binding of peptide ligands.

    In a recent report, the finding that mutation of Asn295 to serine caused constitutive activation of the AT1receptor led to the proposal that an interaction between Asn295 and Asn111 (in the third intramembrane helix) stabilizes the inactive conformation of the AT1 receptor (Balmforthet al., 1997). However, constitutive activation was not observed when Asn295 was replaced by aspartic acid (Perlman et al., 1997), and it was not detectable with the N295A mutant AT1a receptor in this study, despite its reasonably high levels of expression.

    Mutations in the seventh transmembrane helix affect the binding of nonpeptide agonists.

    Initial studies on agonist binding to the AT1 receptor demonstrated that several extracellular residues are essential determinants of Ang II binding (Hjorth et al., 1994). Based on these data, it has been proposed that the peptide binding site of the AT1receptor is located in the extracellular region (Hjorth et al., 1994). However, more recent studies have emphasized the role of the intramembrane helices in the agonist-induced conformational change (Noda et al., 1995, 1996; Hunyady et al.,1996; Inoue et al., 1997; Karnik et al., 1997). His256, in the sixth transmembrane helix, has been reported to interact with the side chain of the carboxyl terminal Phe8 of Ang II (Nodaet al., 1995), and Tyr111 of the AT1receptor has been proposed to interact with the side chain of Tyr4 of Ang II (Noda et al., 1996). These data, as well as recent modeling studies (Underwood et al., 1995; Inoue et al., 1997), point to the importance of the intramembrane binding pocket in the agonist-induced conformational change of the AT1 receptor.

    Asn294 has a role in the agonist-induced conformational change.

    The seventh transmembrane helix has been implicated in several recent studies as the site of initiation of agonist-induced conformational changes in a variety of GPCRs. The retinal chromophore of rhodopsin binds covalently to the seventh transmembrane helix, which has also been implicated in agonist binding to several different GPCRs (Probst et al., 1992; Baldwin, 1993; Strader et al., 1995; Hunyady et al., 1996). More recently, conserved aromatic residues in the seventh transmembrane domain of the same receptor were found to be essential for agonist binding and stimulation of inositol phosphate production (Roth et al., 1997). Rotation of transmembrane helices, which results in a new hydrogen bond between Asp74 of the second transmembrane helix and Tyr292 of the seventh transmembrane helix, has also been proposed as the initial event of AT1 receptor activation (Inoue et al., 1997).

    The polar nature of the amino acid located in the position of Asn294 is a conserved feature of most GPCRs (Probst et al., 1992;Baldwin, 1993). In a published alignment of GPCR sequences, >60% of the listed sequences contain asparagine in the position corresponding to Asn294 of the AT1 receptor, and >90% of the GPCRs have a polar amino acid that can participate in hydrogen bond formation (e.g., serine, threonine, histidine, or arginine) (Probstet al., 1992). In view of the location of Asn294 in the seventh intramembrane helix (Baldwin, 1993; Liu et al.,1995; Mizobe et al., 1996), this residue is unlikely to directly mediate receptor coupling to Gq or other G proteins. It is more probable that this residue is required for an intramolecular interaction that in turn stabilizes the active conformation of the receptor. In recent reports, an interaction between Asn111 and Tyr292 (Groblewski et al., 1997) and/or Asn295 (Balmforth et al., 1997) was proposed to be a major determinant of the inactive conformation of the AT1 receptor. It is possible that an interaction between Asn294 and other amino acids is an additional structural requirement for AT1 receptor activation, similar to the proposed role for the interaction between Tyr292 of the seventh transmembrane helix and Asp74 of the second transmembrane helix (Bihoreau et al., 1993; Marie et al., 1994;Joseph et al., 1995).

    In summary, the present data provide evidence that the binding of nonpeptide agonists to the AT1 receptor depends on residues located in the intramembrane binding pocket. In addition, Asn294 has a crucial role in receptor activation, and Asn295 is a major determinant of the binding of all tested Ang II ligands to the AT1 angiotensin receptor. The complementary functions of these adjacent asparagine residues could provide a molecular basis for the proposed role of the seventh transmembrane helix in transforming agonist binding into the rearrangement of intramembrane helices that is believed to result in receptor activation.

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    Acknowledgments

    We are grateful to Yue Zhang and Katinka Süpeki for expert technical assistance.

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    Footnotes

    • Send reprint requests to: Kevin J. Catt M.D., Ph.D., Endocrinology and Reproduction Research Branch, NICHD, NIH, Building 49, Room 6A-36, Bethesda, MD 20892-4510. E-mail:catt{at}helix.nih.gov

    • This work was supported in part by an International Research Scholar Award from the Howard Hughes Medical Institute (HHMI 75195–541702) and by grants from the Hungarian Ministry of Culture and Education (FKFP-0776/1997) and the Hungarian Ministry of Public Health (ETT 535/96). L.H. is an International Research Scholar of the Howard Hughes Medical Institute.

    • Abbreviations:
      GPCR
      G protein-coupled receptor
      Ang II
      angiotensin II
      InsP2
      inositol bisphosphate
      InsP3
      inositol trisphosphate
      GTPγS
      guanosine 5′-(γ-thio)triphosphate
      BSA
      bovine serum albumin
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      • Received October 13, 1997.
      • Accepted May 7, 1998.
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    1. Molecular Pharmacology August 1, 1998 vol. 54 no. 2 427-434
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