ACCELERATED COMMUNICATION Homologous and Heterologous Phosphorylation of the AT2 Angiotensin Receptor by Protein Kinase C

The angiotensin AT2 receptor is an atypical seven transmembrane domain receptor that is coupled to activation of tyrosine phosphatase and inhibition of MAP kinase, and does not undergo agonist-induced internalization. An investigation of the occurrence and nature of AT2 receptor phosphorylation revealed that phorbol ester-induced activation of protein kinase C (PKC) in HA-AT2 receptor-expressing COS-7 cells caused rapid and specific phosphorylation of a single residue (Ser) located in the cytoplasmic tail of the receptor. Agonist activation of AT2 receptors by angiotensin II (Ang II) also caused rapid PKC-dependent phosphorylation of Ser that was prevented by the AT2 antagonist, PD123177, and by inhibitors of PKC. In cells coexpressing AT1 and AT2 receptors, Ang II-induced phosphorylation of the AT2 receptor was reduced by either PD123177 or the AT1 receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT2 receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation of the AT1 receptor. The latter process may regulate the counteracting effects of AT2 receptors on growth responses to AT1 receptor activation. The superfamily of seven transmembrane domain G protein-coupled receptors (GPCRs) mediates the responses of cells to light, odorants, neurotransmitters, biogenic amines, and numerous hormones. The current view of GPCR function and regulation, which is based largely on studies of the b2-adrenergic receptor (b2-AR), invokes an agonist-dependent change in receptor conformation that allows receptor coupling to cognate G protein(s). This conformational change also promotes receptor phosphorylation by G protein-coupled receptor kinases (GRKs) and/or second messenger-activated kinases. The subsequent binding of b-arrestin proteins desensitizes the receptor by sterically inhibiting its coupling to G proteins, and also mediates its internalization via clathrincoated pits (reviewed in Bohm et al., 1997; Krupnick and Benovic, 1998; Pitcher et al., 1998). Although this model of b2-AR action has been extrapolated to other GPCRs, it does not apply to all of them, and some such receptors use modified or alternative mechanisms of desensitization and internalization, or none at all. For example, the gonadotropin-releasing hormone receptor functionally desensitizes and internalizes very slowly, and does not undergo agonist-induced phosphorylation (Neill et al., 1997). Also, the parathyroid hormone receptor internalizes independently of phosphorylation (Malecz et al., 1998), and endocytosis of the m1, m3, and m4 muscarinic receptors (Lee et al., 1998), and possibly the AT1 angiotensin receptor (AT1-R) (Zhang et al., 1996), appears to be independent of b-arrestins. In contrast to most other GPCRs, the AT2 angiotensin receptor (AT2-R) does not undergo internalization in the presence of its endogenous agonist ligand, Ang II (Hunyady et al., 1994; Hein et al., 1997). Although the AT1-R and AT2-R exhibit high affinity for the octapeptide hormone, Ang II, and both are members of the J.A.O-R. was supported by a Pan American Fellowship (NIH/CONACYT 979004). L.H. was supported in part by an International Research Scholar Award from the Howard Hughes Medical Institute and a Collaborative Research Initiative Grant from the Wellcome Trust (051804/Z/97/Z). S.J. was supported in part by an Alpha Omega Alpha Student Fellowship. ABBREVIATIONS: GPCR, G protein-coupled receptor; AT1-R and AT2-R, types 1 and 2 angiotensin II receptors, respectively; b2-AR, b2adrenergic receptor; BIM, bisindolylmaleimide; DG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; PKC, protein kinase C; PNGase F, peptide N-glycosidase F; SP, staurosporine; TFMS, trifluoromethanesulfonic acid; TPA, 12-O-tetradecanoylphorbol 13-acetate; Ang II, angiotensin II; PAGE, polyacrylamide gel electrophoresis. 0026-895X/00/051156-06$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 5 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 350/861327 Mol Pharmacol 58:1156–1161, 2000 Printed in U.S.A. 1156 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from GPCR superfamily, they share only 32 to 34% amino acid sequence homology (Kambayashi et al., 1993; Mukoyama et al., 1993) and have completely different functions. All of the classical actions of Ang II in the regulation of salt/water balance and blood pressure control are mediated by the Gq-coupled AT1-R. Due to its central role in cardiovascular regulation, many aspects of the structure and function of the AT1-R have been analyzed and elucidated. Although less is known about the functions of the AT2-R, recent studies have shown that its activation can counteract the mitogenic and hypertensive effects mediated via the AT1-R. AT2-R activation exerts antiproliferative and/or apoptotic effects in certain cells (Yamada et al., 1996), and exerts a hypotensive effect in AT1-R-deficient mice (Oliverio et al., 1998). In addition, the wide distribution of the AT2-R in fetal tissues, in contrast to its limited expression in adult tissues, suggests a role for this receptor in developmental processes (de Gasparo and Siragy, 1999). In contrast to the well characterized signal transduction pathways that mediate Ang II actions through the AT1-R, those that are activated by the AT2-R are less well defined. For example, AT2-R activation has been reported to either stimulate (Gohlke et al., 1998) or inhibit (Bottari et al., 1992) cyclic GMP production, and to activate phosphotyrosine phosphatases (such as SHP-1) (Bottari et al., 1992; Kambayashi et al., 1993; Nahmias et al., 1995; Tsuzuki et al., 1996; Bedecs et al., 1997) as well as serine/threonine phosphatase activity (Huang et al., 1996). In addition, there is conflicting evidence about the extent to which the AT2-R can couple to G proteins (Bottari et al., 1991; Kambayashi et al., 1993; Kang et al., 1993; Mukoyama et al., 1993; Zhang and Pratt, 1996). Although the AT1 receptor and many other GPCRs have been found to undergo agonist-induced phosphorylation of their cytoplasmic domains, phosphorylation of the AT2-R has not yet been investigated. In this study, we undertook an analysis of AT2-R phosphorylation to seek insights into the activation and signaling mechanism(s) of this receptor. To this end, the phosphorylation of an epitope-tagged rat AT2-R was investigated in transiently transfected COS-7 cells. Experimental Procedures Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and antibiotic solutions were from Biofluids (Rockville, MD). Ang II, [Sar,Ile]Ang II, and CGP42112 were from Peninsula Laboratories (Belmont, CA). DuP753 and PD123177 were generous gifts from Dr. P. C. Wong (DuPont, Wilmington, DE). I-[Sar,Ile]Ang II and I-[Sar,(4-N3)Phe ]Ang II were from Covance Laboratories (Vienna, VA), and Pi was from ICN (Costa Mesa, CA). Protein A Sepharose was from Oncogene Research Products (Cambridge, MA), and the HA.11 mouse monoclonal antibody was from BAbCo (Berkeley, CA). Peptide N-glycosidase F (PNGase F: E.C. 3.5.1.52) was from Boehringer Mannheim (Indianapolis, IN). Bisindolylmaleimide (BIM) and staurosporine (SP) were from Calbiochem (San Diego, CA). OptiMEM and LipofectAMINE were from Life Technologies, Inc. (Gaithersburg, MD). Trifluoromethanesulfonic acid (TFMS) and 12-O-tetradecanoylphorbol 13-acetate (TPA) were from Sigma (St. Louis, MO). Epitope-Tagging and Mutagenesis of the Rat AT2-R. A HindIII/NsiI fragment of the rat AT2 receptor was subcloned into the eukaryotic expression vector, pcDNAI/Amp (Invitrogen, San Diego, CA), as previously described (Hunyady et al., 1994). The influenza hemagglutinin (HA) epitope (YPYDVPDYA) was inserted after the N-terminal methionine residue using the Mutagene kit (Bio-Rad, Hercules, CA), and its sequence was verified using Sequenase II (Amersham, Arlington Heights, IL). The presence of the epitope tag had no effect on the ligand binding properties of the HA-AT2-R (data not shown). 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, Arlington Heights, IL). Transient Expression of HA-AT2-Rs. COS-7 cells were maintained in DMEM containing 10% (v/v) fetal bovine serum, 100 mg/ml streptomycin, and 100 IU/ml penicillin (COS-7 medium). Cells were seeded at 8 3 10 cells/10-cm dish in COS-7 medium and cultured for 3 days before transfection using 5 ml/dish OptiMEM containing 10 mg/ml LipofectAMINE and the required DNA (1 mg/ml) for 6 h at 37°C. After changing to fresh COS-7 medium, the cells were cultured for an additional 2 days before use. HA-AT2-Rs were photoaffinitylabeled with I-[Sar,(4-N3)Phe ]Ang II as described (Smith et al., 1998a). To quantify the relative phosphorylation of mutant HA-AT2Rs, membrane lysates were normalized to an equal number of HAAT2-Rs (on the basis of Bmax values obtained from radioligand displacement assays using replicate transfected cells) before immunoprecipitation as described (Smith et al., 1998b). HA-AT2-R Phosphorylation Assay. Transfected Cos-7 cells in 10-cm dishes were metabolically labeled for 4 h at 37°C in Pi-free DMEM containing 0.1% (w/v) BSA and 100 mCi/ml Pi as described previously (Smith et al., 1998b). In brief, after three washes in KRH (118 mM NaCl, 2.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM glucose, 20 mM HEPES, pH 7.4), cells were incubated in the same medium for 10 min in a 37°C water bath. Vehicle or 100 nM Ang II was then added for an additional 5 min. After three washes with ice-cold PBS, cells were drained before scraping into lysis buffer (LB2: 50 mM Tris, pH 8.0, 100 mM NaCl, 20 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml soybean trypsin inhibitor, 10 mg/ml pepstatin, 10 mg/ml benzamidine, 1 mM AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride], 1 mM okadaic acid) and probe-sonicated (Sonifier Cell Disruptor; Heat Systems Ultrasonics, Plainview, NY) for 2 3 20 s. After removal of nuclei at 750g, membranes were pre-extracted by the addition of an equal volume of LB2 containing 2 M NaCl and 8 M urea, followed by overnight tumbling at 4°C. The membranes were then collected at 200,000g and solubilized in LB1 [LB2 supplemented with 1% (v/v) NP 40, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS] with Dounce homogenization. After clarification at 14,000g, solubilized membranes were incubated with 2% (v/v) protein A Sepharose for 1 h at 4°C. HA-AT2-Rs were immunoprecipitated from solubilized membrane lysates using the HA.11 monoclonal antibody and 2% (v/v) protein A Sepharose as described (Smith et al., 1998b). Chemical Deglycosylation of HA-AT2-Rs. After washing of the Sepharose-bound immune complexes in LB1 lacking protease inhibitors, P-labeled phospho-HA-AT2-Rs were eluted into 50 ml of buffer containing 2% (v/w) SDS, 5% (v/v) b-mercaptoethanol, and 80 mM Tris (pH 6.8) for 1 h at 48°C. After the addition of ovalbumin as carrier, proteins were precipitated by the addition of ice-cold trichloroacetic acid, collected by centrifugation, washed twice in ice-cold acetone, and dried in a rotary evaporator. Samples were then subjected to chemical deglycosylation using TFMS as previously described (Horvath et al., 1989). After chemical deglycosylation, proteins were redissolved in sample buffer and incubated for 1 h at 48°C before SDS-polyacrylamide gel electrophoresis (PAGE). After drying (Gel-Dry; Novex, San Diego, CA), P-labeled phospho-HA-AT2-Rs were visualized in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).