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A Conserved NPLFY Sequence Contributes to Agonist Binding and Signal Transduction but Is Not an Internalization Signal for the Type 1 Angiotensin II Receptor

1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês

10.1074/jbc.270.28.16602

1083-351X

László Hunyady, Márta Bor, Albert J. Baukal, Tamás Balla, Kevin Catt,

Neuropeptides and Animal Physiology

A conserved NPX2-3Y sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn298 and was reduced by replacement of Pro299 or Tyr302. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar1,Ile8]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe301, Tyr302, and Phe304, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe301 residue contributes significantly to agonist binding, and Asn298 is required for normal receptor activation and signal transduction. A conserved NPX2-3Y sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn298 and was reduced by replacement of Pro299 or Tyr302. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar1,Ile8]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe301, Tyr302, and Phe304, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe301 residue contributes significantly to agonist binding, and Asn298 is required for normal receptor activation and signal transduction. The type 1 (AT1)1 1The abbreviations used are: AT1 receptortype 1 angiotensin II receptorAng IIangiotensin IIInsP2inositol bisphosphateInsP3inositol trisphosphateGTPγSguanosine 5'-3-O-(thio)triphosphate. receptor for the vasoactive peptide Ang II is a member of the family of seven transmembrane domain receptors(1Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 2Catt K.J. Sandberg K. Balla T. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Boca Raton, FL1993: 307-356Google Scholar). The AT1 receptor has been reported to interact with several G proteins, including Gq, Gi, and Go, but its major physiological functions are expressed through Gq-mediated activation of phospholipase C followed by phosphoinositide hydrolysis and Ca2+ signaling(1Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 2Catt K.J. Sandberg K. Balla T. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Boca Raton, FL1993: 307-356Google Scholar, 3Spät A. Enyedi P. Hajnóczky Gy. Hunyady L. Exp. Physiol. 1991; 76: 859-885Crossref PubMed Scopus (77) Google Scholar, 4Catt K.J. Hunyady L. Balla T. J. Bioenerg. Biomembr. 1991; 23: 7-27PubMed Google Scholar, 5Schultz G. Hescheler J. Arzneimittelforschung. 1993; 43: 229-232PubMed Google Scholar). During the last decade, hundreds of G protein-coupled receptors and their subtypes have been cloned and sequenced(6Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (685) Google Scholar, 7Kolakowski Jr., L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). Although these receptors all possess the basic seven-transmembrane structure, the number of highly conserved amino acids shared by the superfamily of G protein-coupled receptors is relatively few(6Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (685) Google Scholar, 8Savarese T.M. Fraser C.M. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (442) Google Scholar, 9Donnelly D. Findlay J.B.C. Blundell T.L. Receptors Channels. 1994; 2: 61-78PubMed Google Scholar). One such conserved motif is the characteristic NPX2-3Y sequence that is located in the seventh transmembrane domain of most receptors, and in the rat smooth muscle AT1a receptor is Asn298-Pro299-Leu300-Phe301-Tyr302 (10Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1171) Google Scholar) (Fig. 1). type 1 angiotensin II receptor angiotensin II inositol bisphosphate inositol trisphosphate guanosine 5'-3-O-(thio)triphosphate. Several G protein-coupled receptor models have been based on the structure of bacteriorhodopsin. This heptahelical membrane protein shares no sequence homology with mammalian G protein-coupled receptors, but it has an identical folding pattern and functional resemblance to mammalian opsins(11Trumpp-Kallmeyer S. Hoflack J. Bruinvels A. Hibert M. J. Med. Chem. 1992; 35: 3448-3462Crossref PubMed Scopus (425) Google Scholar). The recently reported low resolution structural image of bovine rhodopsin (12Schertler G.F.X. Villa C. Henderson R. Nature. 1993; 362: 770-772Crossref PubMed Scopus (714) Google Scholar) has made it possible to further improve these models(9Donnelly D. Findlay J.B.C. Blundell T.L. Receptors Channels. 1994; 2: 61-78PubMed Google Scholar, 13Baldwin J.M. EMBO J. 1993; 12: 1693-1703Crossref PubMed Scopus (886) Google Scholar). A recent model of aminergic G protein-coupled receptors suggests that the NPX2-3Y sequence is ideally placed to receive a signal from agonist-induced conformational changes in the ligand binding region(9Donnelly D. Findlay J.B.C. Blundell T.L. Receptors Channels. 1994; 2: 61-78PubMed Google Scholar, 14Findlay J.B.C. Donnelly D. Bhogal N. Hurrell C. Attwood T.K. Biochem. Soc. Trans. 1993; 21: 869-873Crossref PubMed Scopus (12) Google Scholar). This sequence is in close proximity to the functionally important asparagine-aspartic acid pair located in transmembrane segments 1 and 2 and may participate in important hydrogen bonding interactions. Most of the available models also predict that the highly conserved intramembrane proline residues, which disrupt the α-helical structure of the transmembrane domains, serve as hinges that participate in the agonist-induced conformation change of G protein-coupled receptors(9Donnelly D. Findlay J.B.C. Blundell T.L. Receptors Channels. 1994; 2: 61-78PubMed Google Scholar, 11Trumpp-Kallmeyer S. Hoflack J. Bruinvels A. Hibert M. J. Med. Chem. 1992; 35: 3448-3462Crossref PubMed Scopus (425) Google Scholar). One such proline residue is located within the seventh transmembrane domain in the conserved NPX2-3Y sequence, and this residue (Pro540) of the m3 muscarinic receptor has been found to be important for signal transduction(15Wess J. Nanavati S. Vogel Z. Maggio R. EMBO J. 1993; 12: 331-338Crossref PubMed Scopus (153) Google Scholar). Another interesting feature of the NPX2-3Y sequence is its similarity to the NPXY internalization sequence that was first described in the cytoplasmic segment of the low density lipoprotein receptor(16Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar). In recent studies, the tyrosine residue in this sequence was found to be essential for sequestration of the β-adrenergic receptor (17Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar) but not for the internalization of the gastrin-releasing peptide receptor(18Slice L.W. Wong H.C. Sternini C. Grady E.F. Bunnett N.W. Walsh J.H. J. Biol. Chem. 1994; 269: 21755-21762Abstract Full Text PDF PubMed Google Scholar). The gastrin-releasing peptide receptor and the AT1 receptors, unlike the β-adrenergic receptor, contain an additional aromatic amino acid (Phe321 and Phe301, respectively) in their NPX2-3Y sequences(1Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 18Slice L.W. Wong H.C. Sternini C. Grady E.F. Bunnett N.W. Walsh J.H. J. Biol. Chem. 1994; 269: 21755-21762Abstract Full Text PDF PubMed Google Scholar). The presence of such residue might be important since phenylalanine can substitute for the tyrosine residue in the NPXY internalization sequence of nutrient receptors(16Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar). The present study was performed to analyze the role of the NPX2-3Y sequence in ligand binding, internalization, and signaling of G protein-coupled receptors, utilizing the rat AT1a receptor as a model to evaluate its participation in these critical aspects of receptor function. The cDNA clone (pCa18b) of the rat smooth muscle AT1a receptor subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) was kindly provided by Dr. Kenneth E. Bernstein(10Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1171) Google Scholar). Restriction enzymes were obtained from Boehringer Mannheim 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 mM Ca2+, 1 g/liter bovine serum albumin, and 20 mM HEPES. Lipofectin, lipofectamine and Opti-MEM I were from Life Technologies, Inc. Losartan was a gift from Dr. P. C. Wong (DuPont, Wilmington, DE). 125I-Ang II and [125I-Sar1,Ile8]Ang II were obtained from Hazleton Laboratories (Vienna, VA) or DuPont NEN; [3H]inositol was from Amersham Corp. The rat AT1a receptor cDNA was subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen) as described earlier(19Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar). Mutant rat AT1a receptors were created using the Mutagene kit (Bio-Rad, Hercules, CA), which is based on the method of Kunkel et al.(20Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar). Each mutant contained a silent restriction site to facilitate the screening of colonies. Oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). All mutations were verified by dideoxy sequencing using Sequenase II (U. S. Biochemical Corp.). 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. To determine inositol phosphate responses, internalization kinetics, 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 0.5-ml aliquots of Opti-MEM I containing 8 μg of lipofectamine and 1 μg of plasmid DNA/well, or with increasing amounts of cDNA as indicated in the legend to Fig. 4. The cells were incubated for 5-6 h in this solution, and the medium was replaced with culture medium. For membrane binding studies, 106 cells were grown on 100-mm culture dishes for 3 days. Transfections were performed for 5-6 h using 5 ml of Opti-MEM I containing 16 μg/ml lipofectamine and 10 μg of plasmid DNA. After transfection, the medium was replaced with the culture medium. All experiments were performed 48 h after the initiation of the transfection procedure. To determine the expression level and structural integrity of the mutant receptor, the number of Ang II binding sites was determined by incubating the transfected cells with [125I-Sar1,Ile8]Ang II (0.05-0.1 μCi/sample) and increasing concentrations of unlabeled [Sar1,Ile8]Ang II in Medium 199 (HEPES) for 6 h at 4°C. The cells were washed twice with ice-cold Dulbecco's phosphate-buffered saline, and the radioactivity associated with the cells was measured by γ-spectrometry 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(21Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). 48 h after transfection, the cells were washed and scraped into 1.5 ml of ice-cold 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 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 mM MgCl2, and 20 mM Tris-HCl (pH 7.4)), and the protein content was determined. Binding assays were performed in 0.2 ml of binding buffer supplemented with 2 g/liter bovine serum albumin at 25°C. Each sample contained 0.05-0.1 μCi of 125I-Ang II or [125I-Sar1,Ile8]Ang II, 15-30 μg of crude membranes, and the indicated concentrations of unlabeled [Sar1,Ile8]Ang II, losartan, or Ang II in the presence or absence of 5 μM GTPγS as indicated. After a 90-min incubation at 25°C, the unbound tracer was removed by rapid filtration, and the bound radioactivity was measured by γ-spectrometry. In these experiments, the culture medium was replaced 24 h after transfection with 0.5 ml of inositol-free Dulbecco's modified Eagle's medium containing 1 g/liter bovine serum albumin, 20 μCi/ml [3H]inositol, 2.5% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin as described earlier(19Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar). After 24 h of labeling, the cells were washed twice and incubated in inositol-free modified Medium 199 in the presence of 10 mM LiCl for 30 min at 37°C, and then stimulated with 30 nM Ang II for 20 min or, in the case of F301A, with 1 μM Ang II for 20 min. Incubations were stopped by adding perchloric acid (5% (v/v) final concentration). Inositol phosphates were extracted and analyzed by high performance liquid chromatography as described previously(22Balla T. Sim S.S. Iida T. Choi K.Y. Catt K.J. Rhee S.G. J. Biol. Chem. 1991; 266: 24719-24726Abstract Full Text PDF PubMed Google Scholar). Before each experiment the medium was replaced by Hepes-buffered Medium 199. To determine the internalization kinetics of the mutant and wild-type AT1a receptors 125I-Ang II (0.05-0.1 μCi) was added in the same medium, and the cells were incubated at 37°C for the indicated times. Incubations were stopped by placing the cells on ice and rapidly washing them twice with 1 ml of ice-cold Dulbecco's phosphate-buffered saline. The cells were incubated for 10 min in 0.5 ml of acid wash solution (150 mM NaCl, 50 mM acetic acid) to remove the surface-bound radioligand. The supernatant containing the acid-released radioactivity was collected, and the cells were treated with 0.5 M NaOH and 0.05% SDS to solubilize the acid-resistant (internalized) radioactivity. The radioactivities were measured by γ spectrometry, and the percent of internalized ligand at each time point was calculated from the ratio of the acid-resistant binding to the total (acid-resistant + acid-released) binding. To determine the internalization kinetics of prelabeled receptors, the cells were incubated with 125I-Ang II (0.05-0.1 μCi) in 0.25 ml Medium 199 for 3-4 h at 4°C to permit binding in the absence of receptor internalization. The unbound tracer was removed by washing the cells twice with 1-ml aliquots of ice-cold Dulbecco's phosphate-buffered saline. After addition of 0.5 ml of warm (37°C) Medium 199, the cells were incubated for the indicated times at 37°C to allow internalization to proceed. Incubations were stopped by placing the cells on ice, and the medium containing the tracer released during the incubation was collected and replaced with 0.5 ml of ice-cold acid wash solution. The extracellular (acid-sensitive) and internalized tracer were measured as described above. The total binding was calculated as the sum of the released (into the medium), extracellular (acid-sensitive), and internalized (acid-resistant) radioactivities. The internalized or released radioactivity was expressed as a percent of the total binding at each time point. [Sar1,Ile8]Ang II binding was measured in intact COS-7 cells transfected with mutant or wild-type AT1a receptors to determine the expression level and the functional integrity of these receptors at the plasma membrane. As shown in, all mutant receptors analyzed in this study bound the antagonist radioligand with high affinity. It is interesting to note that all mutants in which Phe301 was replaced (F301A, L300A/F301A, F301A/Y302A, F301A/Y302A/F304A) showed slightly but consistently lower binding affinity than the other mutants or the wild-type receptor. The expression levels of the mutant receptors showed more significant variations. While alanine replacement for Asn298 and Tyr302 had no major effect on receptor expression, mutation of the three inner residues (Pro299, Leu300, and Phe301) reduced expression to approximately one-third of that of the wild-type receptor (Table I). The effect of amino acid replacement on receptor expression appeared to be additive, since L300A/F301A showed further reduction compared with L300A and F301A, and F301A/Y302A and F301A/Y302A/F304A showed progressively reduced expression levels compared with F301A or Y302A.Table I:Parameters of [125I-Sar8,Ile8]Ang II binding for wild-type and mutant AT1a receptors expressed in COS-7 cellsThe data are expressed as means ± S.E. of three independent experiments each performed in duplicate. Open table in a new tab The data are expressed as means ± S.E. of three independent experiments each performed in duplicate. Previous reports showed that the type 1 Ang II receptors can couple to multiple G proteins including Gq, Gi, and Go (1Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar). To evaluate the ability of the AT1a receptor to couple to G proteins, we measured the effect of GTPγS on Ang II binding in COS-7 cell membranes. Addition of GTPγS (5 μM) caused a 3.45 ± 0.38-fold (n = 3) decrease in the affinity of the wild-type receptor for Ang II, indicating that the expressed AT1a receptor is coupled to G proteins. In the presence of the GTP analogue, when the receptor is uncoupled from G proteins, the binding affinities of the N298A, P299A, L300A, and Y302A mutants for Ang II were similar to that of the wild-type receptor (Table II). In the absence of GTPγS, the affinities of the mutants for Ang II were lower than that of the wild-type receptor, suggesting that receptor-G protein interaction is affected in these mutants. The GTPγS-induced change in affinity was most impaired in the N298A and P299A mutant receptors, but it was also evident in the Y302A mutant (Fig. 2). Interestingly, despite its near normal affinity for [Sar1,Ile8]Ang II (Table I), the F301A mutant receptor had significantly reduced affinity for the physiological agonist, Ang II (Table II).Table II:IC50 of 125I-Ang II binding in the absence or presence of 5 μM GTPγS and EC50 of the combined InsP2+ InsP3 responses for wild-type and mutant AT1a receptors expressed in COS-7 cellsThe binding data are expressed as means ± S.E. of three independent experiments each performed in duplicate. The EC50 values are shown as means ± S.E. from independent dose-response curves of two to three experiments. Open table in a new tab The binding data are expressed as means ± S.E. of three independent experiments each performed in duplicate. The EC50 values are shown as means ± S.E. from independent dose-response curves of two to three experiments. Since both the expression levels (Table I) and the agonist affinity (Table II) of the F301A receptor were impaired, it was difficult to accurately characterize this mutant using 125I-Ang II as radioligand. For this reason, [125I-Sar1,Ile8]Ang II was utilized to measure the binding properties of F301A. Consistent with the data obtained in intact cells, the F301A mutant receptor showed only slightly decreased affinity relative to the wild-type receptor when [Sar1,Ile8]Ang II was used to displace the radioligand (IC50: wild-type, 0.53 ± 0.09 nMversus F301A, 0.92 ± 0.05 nM; n = 3) (Fig. 3, upper panel). In contrast, the ability of the agonist ligand (Ang II) to displace [125I-Sar1,Ile8]Ang II was reduced 9.7 ± 0.9-fold in the F301A mutant (IC50: wild-type, 3.0 ± 0.1 nMversus F301A, 29.5 ± 2.6 nM; n = 3) (Fig. 3, middle panel). This selective reduction of Ang II binding affinity was not related to impaired G protein coupling, since the effect of GTPγS on binding was similar to that observed in the wild-type receptor (Fig. 2). The nonpeptide AT1 receptor antagonist losartan inhibited [125I-Sar1,Ile8]Ang II binding to the wild-type receptor with an IC50 of 26.6 ± 1.4 nM (n = 3). However, the affinity of the F301A mutant receptor for losartan was reduced 9.9 ± 2.5-fold (IC50, 263 ± 66 nM; n = 3) (Fig. 3, lower panel), similar to its loss of affinity for the native agonist, Ang II. To determine the ability of the mutant receptors to couple to phospholipase C via Gq and related proteins, we measured the inositol phosphate response of transfected COS-7 cells to Ang II in the presence of LiCl. As reported earlier, under these experimental conditions the major accumulating products of phosphoinositide hydrolysis are InsP2 and InsP3 in AT1a receptor-transfected COS-7 cells(19Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar). Since the expression levels of the mutant AT1a receptors showed significant variations (Table I), the relationship between receptor expression and the amplitude of the maximal inositol phosphate response was determined after transfecting COS-7 cells with increasing amounts of the wild-type AT1a receptor cDNA. Despite the wide range of receptor expression in such cells, there was a linear relationship (r = 0.98) between the measured extracellular receptor sites and the inositol phosphate responses to agonist stimulation (Fig. 4). This finding indicates that valid comparisons between cells expressing mutant AT1a receptors can be made by normalizing their inositol phosphate responses to the number of plasma membrane binding sites (Fig. 5, lower panel). The inositol phosphate responses of cells expressing mutant AT1a receptors were measured after maximal agonist stimulation with 30 nM Ang II. However, 1 μM Ang II was added in studies on the F301A receptor due to its reduced binding affinity for the native agonist. Single alanine replacements in the NPLFY sequence resulted in mutants showing various degrees of reduction of the inositol phosphate responses. The most prominent decrease was detected in the N298A, P299A, and F301A mutants during Ang II stimulation (Fig. 5). After normalization of the data to the receptor expression level, the impaired response of cells transfected with the F301A receptor was attributable to its lower expression level, while the L300A receptor appeared to activate phospholipase C more effectively than the wild-type receptor (Fig. 5, lower panel). The most significant impairment of inositol phosphate signaling was observed in COS-7 cells expressing the N298A receptor, which showed more than 60% reduction of inositol phosphate accumulation. However, the P299A and Y302A receptors also mediated consistently reduced inositol phosphate responses (Fig. 5, lower panel). Since binding studies revealed significant differences in the agonist affinities of the mutant AT1a receptors, a detailed analysis of the dose-dependence of their signaling responses was performed. The EC50 values for inositol phosphate responses of the wild-type and mutant AT1a receptors showed a good correlation with the respective IC50 values for inhibition of radioligand binding by native Ang II (Table II). For example the dose-response curve for Ang II-induced inositol phosphate production of the F301A mutant receptor was shifted to the right compared with that of the wild-type receptor, consistent with the reduced agonist affinity of this mutant (Fig. 6). However, the maximum level of stimulation mediated by the F301A receptor showed no reduction when the data were normalized for the reduced number of binding sites (Fig. 5, lower panel). On the other hand, the higher EC50 values observed in cells expressing the N298A receptor (Fig. 6) and the P299A and Y302A receptors (Table II) were paralleled by impaired G protein-coupling. This is indicated by the reduced maximal response (Fig. 5, lower panel) and decreased GTPγS effect on Ang II binding (Fig. 2) to these mutant receptors. The AT1a receptor expressed in COS-7 cells undergoes rapid agonist-induced internalization, similar to that of the native receptors of smooth muscle, adrenal glomerulosa, and other cell types(2Catt K.J. Sandberg K. Balla T. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Boca Raton, FL1993: 307-356Google Scholar, 19Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar, 23Griendling K.K. Delafontaine P. Rittenhouse S.E. Gimbrone Jr., M.A. Alexander R.W. J. Biol. Chem. 1987; 262: 14555-14562Abstract Full Text PDF PubMed Google Scholar, 24Hunyady L. Merelli F. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1991; 266: 2783-2788Abstract Full Text PDF PubMed Google Scholar). Single alanine replacements in the NPLFY sequence caused relatively minor impairment of the internalization kinetics of the hormone-receptor complex (Fig. 7). While the rates of internalization of the N298A, F301A, and Y302A receptors were somewhat slower than that of the wild-type receptor, all mutant receptors showed rapid agonist-induced internalization, and the quantity of internalized radioligand exceeded that of extracellular binding after a 30-min incubation at 37°C (Fig. 7). In the low density lipoprotein receptor, the tyrosine residue of the NPXY motif can be replaced by phenylalanine with no loss of function of the internalization signal(16Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar). Since the AT1a receptor Tyr302 is preceded by a phenylalanine, the Y302A mutant contains an NPLF sequence that meets the criteria of an internalization signal. To evaluate the possibility that the neighboring phenylalanine residues could substitute for Tyr302 when the latter is replaced with alanine, double (F301A/Y302A) and triple (F301A/Y302A/F304A) alanine replacement mutants were analyzed. Like the F301A mutant receptor, these mutants showed reduced expression levels and slightly reduced antagonist binding (Table II) but exhibited markedly impaired agonist binding (data not shown). However, these mutant receptors underwent rapid agonist-induced endocytosis, indicating that the NPX2-3Y sequence is not a major determinant of the internalization of the AT1a receptor (Fig. 8). To further analyze the role of Tyr302 in the endocytosis of AT1a receptors, internalization kinetics were measured in cells prelabeled with 125I-Ang II. Prelabeling was performed at 4°C to prevent internalization of the radioligand as described under "Experimental Procedures." After warming the cells to 37°C, more than 60% of the tracer bound to the wild-type receptor internalized within 5 min (Fig. 9, upper panel) similar to the previously reported rapid kinetics of endogenous AT1 receptors(23Griendling K.K. Delafontaine P. Rittenhouse S.E. Gimbrone Jr., M.A. Alexander R.W. J. Biol. Chem. 1987; 262: 14555-14562Abstract Full Text PDF PubMed Google Scholar, 24Hunyady L. Merelli F. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1991; 266: 2783-2788Abstract Full Text PDF PubMed Google Scholar). At the same time (5 min), less than 20% of the radioactivity was released into the medium (Fig. 9, lower panel), indicating that dissociation of the agonist from the receptor is relatively slow in comparison with the rapid kinetics of the internalization process. The release of bound and internalized radioactivity into the incubation medium exhibited biphasic kinetics. As shown for the wild-type receptor (Fig. 9, lower panel), the initial phase of release, which is largely due to dissociation of the surface-bound ligand, began to plateau by 5 min, reflecting the concomitant decrease in