Agonist-induced Signaling, Desensitization, and Internalization of a Phosphorylation-deficient AT1A Angiotensin Receptor
2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês
10.1074/jbc.m106368200
ISSN1083-351X
AutoresJ. Alberto Olivares‐Reyes, Roger Smith, László Hunyady, Bukhtiar H. Shah, Kevin Catt,
Tópico(s)Neuropeptides and Animal Physiology
ResumoAn analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by ∼40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by ∼30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor. An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by ∼40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by ∼30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor. G protein-coupled receptor type 1 angiotensin receptor type 2 angiotensin receptor β2-adrenergic receptor bisindolylmaleimide Asp236-Asp237mutant type 1 angiotensin receptor Dulbecco's modified Eagle's medium fetal bovine serum G protein-coupled receptor kinase dominant negative mutant of GRK2 hemagglutinin protein kinase C peptideN-glycosidase F angiotensin II bovine serum albumin phosphate-buffered saline polyacrylamide gel electrophoresis The most intensively studied members of the superfamily of G protein-coupled receptors (GPCRs)1 are rhodopsin and the β2-adrenergic receptor (β2-AR). Following agonist activation, Gs-mediated signaling by the β2-AR is rapidly attenuated (or desensitized) and the receptor is internalized (or sequestered) into cells. In many GPCRs, these events result from phosphorylation of the agonist-activated receptors on serine/threonine residues by G protein-coupled receptor kinases (GRKs) and the consequent binding of β-arrestin proteins. Receptor-bound β-arrestins not only uncouple the β2-AR from Gs and initiate desensitization, but also act as molecular adaptors in the endocytosis of β2-ARs via clathrin-coated pits and promote intracellular signaling to mitogen-activated protein kinase cascades (reviewed in Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 3Hunyady L. Catt K.J. Clark A.J. Gaborik Z. Regul. Pept. 2000; 91: 29-44Crossref PubMed Scopus (98) Google Scholar, 4Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Agonist-induced desensitization occurs in almost all GPCRs, and is mediated by phosphorylation of the receptor by at least two classes of serine/threonine kinase: GRKs and second messenger-activated kinases, usually protein kinase A or C. Agonist-induced phosphorylation has been demonstrated in numerous GPCRs including the β2-AR, m1 (5Haga K. Kameyama K. Haga T. Kikkawa U. Shiozaki K. Uchiyama H. J. Biol. Chem. 1996; 271: 2776-2782Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and m2 (6Richardson R.M. Kim C. Benovic J.L. Hosey M.M. J. Biol. Chem. 1993; 268: 13650-13656Abstract Full Text PDF PubMed Google Scholar) muscarinic, substance P (7Kwatra M.M. Schwinn D.A. Schreurs J. Blank J.L. Kim C.M. Benovic J.L. Krause J.E. Caron M.J. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 9161-9164Abstract Full Text PDF PubMed Google Scholar), somatostatin (8Hipkin R.W. Friedman J. Clark R.B. Eppler M. Schonbrunn A. J. Biol. Chem. 1997; 272: 13869-13876Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), δ-opioid (9Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar), endothelin (10Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. 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Chem. 2001; 276: 4917-4922Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and the μ opioid receptor (29Pak Y. O'Dowd B.F. George S.R. J. Biol. Chem. 1997; 272: 24961-24965Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 30Deng H.B. Yu Y. Pak Y. O'Dowd B.F. George S.R. Surratt C.K. Uhl G.R. Wang J.B. Biochemistry. 2000; 39: 5492-5499Crossref PubMed Scopus (55) Google Scholar) have shown that neighboring acidic residues are necessary for GRK-mediated phosphorylation. The major agonist-induced phosphorylation sites of the rat AT1A-R are located in a serine/threonine-rich segment (Ser326–Thr338) of the receptor's cytoplasmic tail (15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar, 16Thomas W.G. Motel T.J. Kule C.E. Karoor V. Baker K.M. Mol. Endocrinol. 1998; 12: 1513-1524Crossref PubMed Scopus (84) Google Scholar). However, the only potentially relevant acidic residues within the intracellular domains of the AT1-R are Asp236 and Asp237, and these are located in the receptor's third intracellular loop rather than adjacent to the phosphorylation sites in the COOH-terminal cytoplasmic domain (31Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1168) Google Scholar). Earlier studies have shown that individual mutations of these aspartate residues have no major effect on inositol phosphate signaling and internalization of the AT1 receptor (32Zhang M. Zhao X. Chen H.-C. Catt K.J. Hunyady L. J. Biol. Chem. 2000; 275: 15782-15788Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). However, in the present study, a more detailed analysis of this diacidic motif has demonstrated its importance in GRK-mediated phosphorylation of the receptor, and has raised questions about the general role of these kinases in the regulation of desensitization and endocytosis of GPCRs. This investigation of the functional importance of the diacidic motif has indicated its requirement in agonist-induced receptor phosphorylation of the AT1-R and has also suggested that the majority of such phosphorylation is not essential for receptor desensitization and internalization. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from American Type Culture Collection. Phosphate-free DMEM, inositol-free DMEM, trypsin, antibiotic solutions, OptiMEM, and LipofectAMINE were from Life Technologies, Inc. Medium 199 was from Biofluids (Rockville, MD). Angiotensin II was from Peninsula Laboratories (Belmont, CA).125I-[Sar1,Ile8]Ang II and125I-Ang II were from Covance Laboratories (Vienna, VA),myo-[2-3H]inositol was fromAmersham Pharmacia Biotech, and 32Pi was from ICN (Costa Mesa, CA). Protein A-agarose and bisindolylmaleimide I were all from Calbiochem (San Diego, CA). Peptide N-glycosidase F (N-glycanase) F (PNGase F; EC 3.5.1.52) was from Roche Molecular Biochemicals and from Glyco (Heyford, United Kingdom), and the HA.11 mouse monoclonal antibody was from BAbCo (Berkeley, CA). The influenza HA epitope (YPYDVPDYA) was inserted after the codons of the amino-terminal first two amino acids (MA) into the cDNA of the rat AT1A receptor subcloned into pcDNA3.1 (Invitrogen, San Diego, CA) as described previously (14Smith R.D. Baukal A.J. Zolyomi A. Gaborik Z. Hunyady L. Sun L. Zhang M. Chen H.-C. Catt K.J. Mol. Endocrinol. 1998; 12: 634-644Crossref PubMed Scopus (41) Google Scholar). The expression vector pcDNA I-GRK2 dominant negative mutant (GRK2K220M) was kindly donated by Dr. S. S. G. Ferguson (33Ferguson S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Site-directed mutagenesis was achieved using the Quick Change kit (Stratagene, La Jolla, CA), and mutant sequences were verified by dideoxy sequencing using Thermosequenase (Amersham Pharmacia Biotech). COS-7 cells (American Type Culture Collection) were grown at 37 °C in a humidified atmosphere of 95% air, 5% CO2, in DMEM supplemented with 10% FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin (COS-7 medium). Cells were seeded at 8 × 105 cells/10-cm dish in COS-7 medium and cultured for 3 days prior to transfection using 5 ml/dish OptiMEM containing 10 μg/ml LipofectAMINE and the required pcDNA3.1 expression vectors for HA-AT1ARs (wild-type and DD mutants) alone (5 μg/100-mm dish) or together with the pcDNA I-GRK2K220M (3.75 and 2.5 μg DNA/100-mm dish, respectively) for 6 h at 37 °C. After changing to fresh COS-7 medium, the cells were cultured for another 2 days prior to use. Binding of 125I-[Sar1,Ile8]Ang II to intact cells was determined as described previously (34Hunyady L. Zhang M. Jagadeesh G. Bor M. Balla T. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10040-10045Crossref PubMed Scopus (36) Google Scholar). Transfected COS-7 cells in 10-cm dishes were metabolically labeled for 4 h at 37 °C in 5 ml of Pi-free DMEM containing 0.1% (w/v) BSA and 100 μCi/ml 32Pi. After three washes in KRH (118 mm NaCl, 2.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2, 10 mm glucose, 0.1% (w/v) BSA, 20 mm Hepes, pH 7.4), cells were incubated in the same medium for 10 min in a 37 °C water bath and Ang II (100 nm) was added for another 5 min. After three washes with ice-cold PBS, membrane lysates were prepared, extracted with salt/urea, and solubilized prior to deglycosylation with PNGase F as described (14Smith R.D. Baukal A.J. Zolyomi A. Gaborik Z. Hunyady L. Sun L. Zhang M. Chen H.-C. Catt K.J. Mol. Endocrinol. 1998; 12: 634-644Crossref PubMed Scopus (41) Google Scholar, 15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar). Following immunoprecipitation with the HA.11 antibody and protein A-Sepharose, 32P-labeled phospho-HA-AT1A-Rs were eluted into sample buffer for 1 h at 48 °C and resolved by SDS-PAGE (8–16% gradient resolving gel) prior to visualization in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). In order to quantify the relative phosphorylation of mutant HA-AT1A-Rs, membrane lysates were normalized to an equal number of HA-AT1A-Rs prior to immunoprecipitation. COS-7 cells from replicate 10-cm dishes were detached by trypsinization 24 h after transfection, reseeded into 24-well plates, cultured for another 24 h, and subjected to radioligand binding competition assay using125I-[Sar1,Ile8]Ang II.Bmax values were obtained from Scatchard analysis of the binding data using the LIGAND program. 125I-Ang II was added in serum-free Medium 199 containing 1 g/liter bovine serum albumin to transfected COS-7 cells in 24-well plates maintained at 37 °C for the required times. Incubations were stopped by rapid washing with ice-cold PBS, and acid-released and acid-resistant radioactivities were separated and measured by γ-spectrometry as described previously (35Hunyady L. Bor M. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 31378-31382Abstract Full Text PDF PubMed Google Scholar). The percentage of internalized ligand at each time point was calculated from the ratio of the acid-resistant specific binding to the total (acid-released + acid-resistant) specific binding. Nonspecific binding was determined in the presence of an excess (10 μm) of unlabeled [Sar1,Ile8]Ang II. Transfected COS-7 cells in 24-well plates were labeled by overnight incubation in inositol-free DMEM containing 0.1% (w/v) BSA, 2.5% (v/v) FBS, antibiotics, and 20 μCi/mlmyo-[2-3H]inositol. After washing and preincubation with 10 mm LiCl for 30 min, 100 nm Ang II was added for another 30 min. Inositol phosphates were extracted as described (36Hunyady L. Ji H. Jagadeesh G. Zhang M. Gaborik Z. Mihalik B. Catt K.J. Mol. Pharmacol. 1998; 54: 427-434Crossref PubMed Scopus (44) Google Scholar) and applied to Bio-Rad AG 1-X8 columns. After washing three times with water and twice with 0.2m ammonium formate, 0.1 m formic acid, the combined [3H]inositol bisphosphate + [3H]inositol trisphosphate fractions were eluted with 1m ammonium formate, 0.1 m formic acid, and radioactivities were determined by liquid scintillation counting. At the expression levels used in this study, there was a linear relationship between the number of cell surface receptors and the magnitude of agonist-stimulated [3H]inositol phosphate production (37Hunyady L. Bor M. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1995; 270: 16602-16609Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Desensitization experiments were performed as described previously (13Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Labeled COS-7 cells expressing the indicated receptors were preincubated in medium alone or with 100 nmAng II for 3 min and washed with PBS before the treatment with 150 mm NaCl, 50 mm glycine, pH 3, for 30 s at 4 °C. After washing three times with ice-cold PBS, the cells were incubated for 15 min in medium containing 10 mm LiCl alone or with 100 nm Ang II. [3H]Inositol phosphates were extracted and measured as described above. Transfected COS-7 cells in 10-cm dishes were grown for 1 day after transfection and reseeded in six-well plates. Cells were serum-starved for 24 h before treatment with 100 nm Ang II for the indicated time. After agonist stimulation at 37 °C, medium was aspirated and cells were washed twice with ice-cold PBS, then lysed in 100 μl of Laemmli sample buffer. The samples were briefly sonicated, heated at 95 °C for 5 min, and centrifuged for 5 min. The supernatants were electrophoresed on SDS-PAGE (8–16%) gradient gels and transferred to polyvinylidene difluoride nylon membranes. GRK2 or phosphorylated ERKl and ERK2 were detected by using a polyclonal rabbit anti-GRK2 antibody (0.5 μg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) or a monoclonal mouse phospho-ERK1/2-specific antibody (1:2000 dilution) (Cell Signaling Technology, Beverly, MA), respectively. Blots were probed with a 1:3000 dilution of anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies and visualized by using by ECL (enhanced chemiluminescence reagent), then quantified by scanning laser densitometry (Amersham Pharmacia Biotech). Substitution of both residues of the diacidic motif (Asp236-Asp237) located at the carboxyl-terminal end of the third intracellular loop (31Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1168) Google Scholar) with either alanine (DD-AA) or asparagine (DD-NN) reduced the expression of the mutant receptors below that of the wild-type AT1 receptor (Table I). However, theKd values of these and other relevant mutant receptors were similar to those of the wild-type receptor, demonstrating that the Asp236-Asp237 motif is not required for high affinity ligand binding to the AT1A-R. In subsequent studies, the relative degrees of agonist-induced phosphorylation of mutant receptors were determined using solubilized membrane lysates normalized to an equal number of receptors, as calculated from Bmax values derived from radioligand binding assays in replicate transfected cells prior to immunoprecipitation.Table IBinding parameters of mutant HA-AT1A-RsReceptorKdBmaxnm% WTWT1.6 ± 0.1100DD-AA1.5 ± 0.252 ± 2DD-NN1.5 ± 0.261 ± 2DD-AD1.7 ± 0.283 ± 2DD-DA1.7 ± 0.240 ± 3DD-ED1.5 ± 0.194 ± 5DD-DE1.6 ± 0.188 ± 5DD-EE1.8 ± 0.281 ± 2Δ3351.6 ± 0.185 ± 4Intact COS-7 cells expressing the indicated receptors were subjected to radioligand binding competition assays for 2 h at 22 °C using125I-[Sar1,Ile8]Ang II, andKd and Bmax values were calculated using the LIGAND program. The Bmax value for the wild-type (WT) receptor was 1.7 ± 0.4 pmol/mg of protein. The data represent mean values (± S.E.) from six to eight independent experiments. Open table in a new tab Intact COS-7 cells expressing the indicated receptors were subjected to radioligand binding competition assays for 2 h at 22 °C using125I-[Sar1,Ile8]Ang II, andKd and Bmax values were calculated using the LIGAND program. The Bmax value for the wild-type (WT) receptor was 1.7 ± 0.4 pmol/mg of protein. The data represent mean values (± S.E.) from six to eight independent experiments. The phosphorylated HA-AT1A-R extracted from Ang II-stimulated COS-7 cells migrates as a diffuse band ofMr 85,000–145,000 in SDS-PAGE (14Smith R.D. Baukal A.J. Zolyomi A. Gaborik Z. Hunyady L. Sun L. Zhang M. Chen H.-C. Catt K.J. Mol. Endocrinol. 1998; 12: 634-644Crossref PubMed Scopus (41) Google Scholar, 15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar). This broad migration pattern, which is largely due to variable degrees of receptor glycosylation and possibly the presence of comigrating phosphoproteins, interferes with the quantitation of the native phospho-HA-AT1A-Rs. For this reason, the solubilized phosphoreceptors were subjected to enzymatic deglycosylation with PNGase F prior to SDS-PAGE analysis. The deglycosylated phospho-HA-AT1A-R runs as a discrete doublet ofMr ∼40,000, consistent with the predicted molecular mass (41 kDa) of the receptor, and is separate from extraneous phosphoproteins (15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar). Under these conditions, minimal basal phosphorylation of the wild-type HA-AT1A-R, or of the two DD mutant receptors, was observed in control cells (Fig.1A). However, exposure of wild-type HA-AT1A-R-expressing COS-7 cells to 100 nm Ang II for 5 min caused prominent phosphorylation of the receptor (Fig. 1A). In contrast, substitution of both Asp236 and Asp237 with alanine (DD-AA) or asparagine (DD-NN) reduced agonist-induced receptor phosphorylation by >60% (Fig. 1B). In principle, mutation of Asp236 and/or Asp237 to alanine or asparagine might "lock" the HA-AT1A-R into a conformation that is incapable of activation in response to agonist. Such inactive receptors would not only fail to isomerize to a conformation that permits phosphorylation, but could also be unable to transduce intracellular signals. However, exposure of DD-AA- or DD-NN-expressing COS-7 cells to Ang II caused a significant increase in [3H]inositol phosphate production (Fig.2A). Furthermore, when Ang II-stimulated increases in [3H]inositol phosphate accumulation were normalized to an equal number of receptors, there was no reduction in the responses of the DD-AA- and DD-NN-expressing cells (Fig. 2B). In addition, Ang II-induced ERK activation, normalized to an equal degree of receptor expression, was also unaffected by the DD mutations. As shown in Fig. 2C, no differences in ERK phosphorylation were observed in COS-7 cells expressing wild-type or DD mutant AT1A receptors after 5 min of stimulation with 100 nm Ang II. These data indicate that the agonist-stimulated DD-AA and DD-NN receptors can isomerize to an active conformation with full efficacy for coupling to Gq/11 signaling and activation of the ERK phosphorylation cascade. Furthermore, since the magnitudes of Ang II-stimulated [3H]inositol phosphate and ERK responses mediated by the DD-AA and DD-NN receptors did not exceed that mediated by the wild-type HA-AT1A-R, phosphorylation may not be essential for the desensitization mechanism(s) that normally limit signaling by the AT1 receptor (15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar). In this context, we compared the magnitudes of Ang II-stimulated [3H]inositol phosphate production mediated by the wild-type and the DD mutants with that mediated by a Δ335 truncated AT1 receptor. As observed previously (15Smith R.D. Hunyady L. Olivares-Reyes J.A. Mihalik B. Jayadev S. Catt K.J. Mol. Pharmacol. 1998; 54: 935-941Crossref PubMed Scopus (66) Google Scholar), Ang II-induced intracellular signaling by the truncated AT1A-R was increased, an effect attributable to reduced desensitization of the hypophosphorylated receptor. The ability of the agonist-activated Δ335 receptor to stimulate inositol phosphate production was much greater than those of the wild-type and DD mutant receptors (Fig. 2, A and B), as was its effect on ERK signaling (data not shown). The capacity of the mutant receptors to undergo agonist-induced desensitization was evaluated (13Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) by pretreatment for 3 min with 100 nm Ang II, which significantly reduced the subsequent maximal Ang II-stimulated inositol phosphate production as compared with non-treated control cells expressing the wild-type receptor (Fig.3A). Attenuation of inositol phosphate generation was also observed when the DD mutant receptors were pretreated with 100 nm Ang II (Fig. 3A). Under similar conditions a small reduction was observed for the Δ335 receptor and was insignificant after normalization for receptor expression (Fig. 3B). In view of the apparent difference in the role that phosphorylation plays in the function of the HA-AT1A-R compared with that of the β2-AR, we also examined the internalization kinetics of the DD-AA and DD-NN receptors in COS-7 cells. Like the [3H] inositol phosphate and ERK responses mediated by these receptors, there was little difference in the rate and magnitude of 125I-Ang II internalization in DD-AA- or DD-NN-expressing cells compared with those expressing the wild-type receptor (Fig. 4). Hence, in contrast to the β2-AR, the majority of the agonist-induced HA-AT1A-R phosphorylation does not appear to be required for its internalization following Ang II stimulation. An evaluation of the structural requirements of the Asp236-Asp237 motif for optimal AT1A-R phosphorylation and signaling responses showed that substitution of either acidic residue with alanine (DD-AD, DD-DA) or glutamic acid (DD-DE, DD-ED) had little effect on Ang II-induced AT1A-R phosphorylation (Fig.5A). Replacement of both Asp236 and Asp237 with glutamic acid residues (DD-EE) likewise did not impair Ang II-induced HA-AT1A-R phosphorylation. Consistent with the normal signaling of the DD-AA and DD-NN receptors (Fig. 2A), the [3H]inositol phosphate responses mediated by the DD-AD, DD-DA, DD-DE, DD-ED, and DD-EE receptors were not significantly reduced (Fig. 5B). Similarly, the rate and magnitude of 125I-Ang II internalization by the mutant receptors were the same as those of the wild-type receptor (Fig. 5C). Taken together, these data indicate that a single acidic residue at position 236 or 237 is necessary for
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