Introduction

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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 ₂-adrenergic receptor (₂-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 -arrestin proteins desensitizes the receptor by sterically inhibiting its coupling to G proteins, and also mediates its internalization via clathrin-coated pits (reviewed in Bohm et al., 1997; Krupnick and Benovic, 1998; Pitcher et al., 1998). Although this model of ₂-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 -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 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 G_q-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

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Materials. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and antibiotic solutions were from Biofluids (Rockville, MD). Ang II, [Sar¹,Ile⁸]Ang II, and CGP42112 were from Peninsula Laboratories (Belmont, CA). DuP753 and PD123177 were generous gifts from Dr. P. C. Wong (DuPont, Wilmington, DE). ¹²⁵I-[Sar¹,Ile⁸]Ang II and ¹²⁵I-[Sar¹,(4-N₃)Phe⁸]Ang II were from Covance Laboratories (Vienna, VA), and ³²P_i 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 µg/ml streptomycin, and 100 IU/ml penicillin (COS-7 medium). Cells were seeded at 8 × 10⁵ cells/10-cm dish in COS-7 medium and cultured for 3 days before transfection using 5 ml/dish OptiMEM containing 10 µg/ml LipofectAMINE and the required DNA (1 µg/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 photoaffinity-labeled with ¹²⁵I-[Sar¹,(4-N₃)Phe⁸]Ang II as described (Smith et al., 1998a). To quantify the relative phosphorylation of mutant HA-AT₂-Rs, membrane lysates were normalized to an equal number of HA-AT₂-Rs (on the basis of B_max 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 P_i-free DMEM containing 0.1% (w/v) BSA and 100 µCi/ml ³²P_i 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 (LB: 50 mM Tris, pH 8.0, 100 mM NaCl, 20 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin, 10 µg/ml benzamidine, 1 mM AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride], 1 µM okadaic acid) and probe-sonicated (Sonifier Cell Disruptor; Heat Systems Ultrasonics, Plainview, NY) for 2 × 20 s. After removal of nuclei at 750g, membranes were pre-extracted by the addition of an equal volume of LB 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 LB+ [LB 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 LB+ lacking protease inhibitors, ³²P-labeled phospho-HA-AT₂-Rs were eluted into 50 µl of buffer containing 2% (v/w) SDS, 5% (v/v) -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), ³²P-labeled phospho-HA-AT₂-Rs were visualized in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

	Results

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Enzymatic Deglycosylation of the Phospho-HA-AT₂-R. Like the rat AT_1a-R (Smith et al., 1998a), the photoaffinity-labeled HA-tagged rat AT₂-R (HA-AT₂-R) expressed in COS-7 cells migrated as a diffuse high-molecular-weight band in SDS-PAGE (Figs. 1 and 2), probably as a result of heterogeneous receptor glycosylation. Treatment of HA-AT₂-R-expressing COS-7 cells with TPA to activate PKC caused a concentration-dependent increase in the phosphorylation of proteins that comigrated as a broad high-molecular-weight band with the photoaffinity-labeled receptor (Fig. 1A). To determine whether these represent the phosphorylated HA-AT₂-R, the photoaffinity-labeled and putative phospho-HA-AT₂-Rs were treated with PNGase F (Lemp et al., 1990), which completely deglycosylates the HA-AT_1a-R (Smith et al., 1998b; Jayadev et al., 1999), before immunoprecipitation. However, overnight treatment with PNGase F (10 U/ml) deglycosylated only a minority of the AT₂-R receptor component, as indicated by its migration in SDS-PAGE to the predicted M_r of the 41-kDa nonglycosylated HA-AT₂-R polypeptide (Kambayashi et al., 1993; Mukoyama et al., 1993). Most of the receptors appeared as a diffuse band with migration intermediate between the fully glycosylated and completely deglycosylated forms (Fig. 1, B and C). Even after overnight incubation with a high concentration (50 U/ml) of PNGase F (not shown), or with 10 U/ml PNGase F for up to 72 h (Fig. 1C), there was no appreciable increase in the proportion of the completely deglycosylated receptor.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Deglycosylation of the phospho-HA-AT2-R. A, membrane lysates prepared from 32P-labeled HA-AT2-R-expressing COS-7 cells were exposed to the indicated concentrations of TPA for 5 min and subjected to immunoprecipitation with the anti-HA antibody. B, membrane lysates from 32P-labeled cells exposed to vehicle (C) or 200 nM TPA (T) for 5 min were incubated overnight in the presence or absence of 10 U/ml PNGase F (P) as indicated before immunoprecipitation with the anti-HA antibody. Membrane lysates from photoaffinity-labeled cells (Az) were incubated without () or with (+) 10 U/ml PNGase F (P) before immunoprecipitation with the anti-HA antibody. C, membrane lysates from photoaffinity-labeled cells were incubated for the indicated times with 10 U/ml PNGase F before immunoprecipitation with the anti-HA antibody. D, membrane lysates prepared from photoaffinity-labeled cells were incubated overnight with 10 U/ml PNGase F (P), or 1 h with TFMS as indicated. E, membrane lysates prepared from photoaffinity-labeled cells were incubated for the indicated times with TFMS. F, membrane lysates prepared from 32P-labeled cells exposed to 200 nM TPA for 5 min were subjected to immunoprecipitation with the anti-HA antibody before treatment for the indicated times with TFMS. In each panel, precipitated proteins were resolved by SDS-PAGE. Az, 125I-[Sar1,(4-N3)Phe8]Ang II photoaffinity-labeled HA-AT2-Rs. Results in this and subsequent figures are representative of data obtained from at least three independent experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Agonist-induced phosphorylation of the HA-AT2-R. A, HA-AT2-R-expressing COS-7 cells were exposed to vehicle (C), 200 nM TPA (T), 100 nM Ang II (A), or 100 nM CGP42112 (CG) for 5 min as indicated. B, 32P-labeled HA-AT2-Rs immunoprecipitated from cells exposed to the indicated concentrations of Ang II for 5 min or pretreated for 10 min with 10 µM PD123177 (PD), before the addition of 10 µM Ang II for an additional 5 min, were treated with TFMS for 5 min. Immunoprecipitated phospho-HA-AT2-Rs were treated with TFMS for 5 min before SDS-PAGE. Az, photoaffinity-labeled HA-AT2-Rs.

Chemical Deglycosylation of the Phospho-HA-AT₂-R. In contrast to PNGase F, treatment of the immunoprecipitated photoaffinity-labeled HA-AT₂-R with TFMS for 60 to 90 min gave rise solely to the completely deglycosylated receptor, and no intermediate forms were present (Fig. 1D). The intensity of the deglycosylated TFMS-treated photoaffinity-labeled receptor was considerably less than that of the untreated receptor. However, TMSF treatment of membranes from photoaffinity-labeled HA-AT₂-R-expressing COS-7 cells for increasing times before immunoblotting with the anti-HA antibody did not change the intensity of the immunoreactive deglycosylated HA-AT₂-R band, whereas the intensity of photoaffinity labeling decreased markedly (data not shown). These findings suggest that prolonged exposure of photoaffinity-labeled HA-AT₂-Rs to TFMS causes loss of the coupled photoaffinity ligand, possibly due to cleavage of its covalent attachment to the receptors' carbohydrate residues.

Agonist-Induced Phosphorylation of the HA-AT₂-R. Ang II-stimulated cells showed an increase in receptor phosphorylation that was similar to that induced by TPA (Fig. 2A), whereas the partial AT₂-R agonist, CGP42112, caused a much smaller increase in phosphorylation. HA-AT₂-R phosphorylation showed a progressive elevation with increasing agonist concentrations and reached a maximum at around 10 nM Ang II (Fig. 2B). Pretreatment with the specific AT₂-R antagonist, PD123177, abolished the HA-AT₂-R phosphorylation observed after 5 min stimulation with 10 µM Ang II (Fig. 2B), indicating the specificity of the agonist-stimulated HA-AT₂-R phosphorylation.

Site(s) of Phosphorylation of the HA-AT₂-R by PKC. The rat AT₂-R contains three residues (Ser¹⁵², Ser³⁴⁸, and Ser³⁵⁴) that are situated within consensus sequences (Ser-Xaa-Lys/Arg) for phosphorylation by PKC (Pearson and Kemp, 1991). We next determined whether the HA-AT₂-R is phosphorylated on one or more of these residues following TPA or Ang II treatment of cells expressing mutant HA-AT₂ receptors with alanine replacements at each of the three candidate serine residues. Scatchard analysis of ¹²⁵I-[Sar¹,Ile⁸]Ang II displacement data from five independent experiments revealed similar dissociation constants for the wild-type and mutant receptors (K_d, 0.65 ± 0.12 nM for wild- type; 0.55 ± 0.08 nM for S152A; 0.66 ± 0.11 nM for S348A; and 0.84 ± 0.11 nM for S354A). The expression levels of the mutant receptors at the cell surface were not significantly different from that of the wild-type receptor (B_max, 2.3 ± 0.4 pmol/mg of protein for wild-type; 2 ± 0.2 pmol/mg of protein for S152A; 2.2 ± 0.1 pmol/mg of protein for S348A; and 2.7 ± 0.2 pmol/mg of protein for S354A). Nevertheless, B_max values obtained from Scatchard analysis of ¹²⁵I-[Sar¹,Ile⁸]Ang II binding to intact replicate transfected COS-7 cells were routinely used to normalize ³²P-labeled membrane lysates to an equal number of HA-AT₂-Rs before immunoprecipitation, to assess the relative degree of phosphorylation of each mutant receptor.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Protein kinase C-mediated phosphorylation of wild-type and mutant HA-AT2-Rs. 32P-Labeled COS-7 cells expressing the indicated HA-AT2-Rs (which were normalized to an equal number of receptors as required), were exposed to vehicle or 200 nM TPA (A) or 100 nM Ang II (B) for 5 min as indicated. C, wild-type HA-AT2-R-expressing cells were pretreated for 10 min with vehicle or 1 µM BIM before the addition of vehicle or 200 nM TPA for an additional 5 min as indicated. D, wild-type HA-AT2-R-expressing cells were pretreated for 10 min with vehicle, 1 µM BIM, or 500 nM SP before the addition of vehicle or 100 nM Ang II for an additional 5 min as indicated. In each panel, phospho-HA-AT2-Rs were immunoprecipitated from membrane lysates (which were normalized to an equal number of receptors as required) and treated with TFMS for 5 min before SDS-PAGE. Az, photoaffinity-labeled HA-AT2-Rs.

Heterologous Ligand-Mediated Phosphorylation of the HA-AT₂-R via the AT_1a-R. Because the HA-AT₂-R can be phosphorylated in response to PKC activation by TPA, we determined whether it was also phosphorylated during heterologous ligand-mediated activation of PKC. For this purpose, COS-7 cells were cotransfected with both the HA-AT₂-R and the wild-type (non-HA-tagged) G_q-coupled rat AT_1a-R. In preliminary experiments, photoaffinity-labeled AT_1a receptors were not immunoprecipitated by the anti-HA antibody (data not shown). Exposure of the cotransfected cells to 100 nM Ang II caused prominent phosphorylation of the HA-AT₂-R (Fig. 4A) that was inhibited by preincubation of the cells with 1 µM BIM or 500 nM SP (Fig. 4B). Receptor phosphorylation was reduced by preincubation with either 10 µM PD123177 or 10 µM Dup753, an AT₁-R-specific antagonist (Chiu et al., 1989), and was abolished by combined treatment with both antagonists (Fig. 4A). Hence, the HA-AT₂-R can be phosphorylated by ligand-mediated activation of PKC via the AT_1a-R, as well as directly during agonist activation.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Heterologous phosphorylation of the HA-AT2-R via the rat AT1a-R. A, COS-7 cells coexpressing the HA-AT2-R and the (non-HA-tagged) rat AT1a-R were pretreated for 10 min with vehicle, 10 µM PD123177 (PD), 10 µM DuP753 (DuP), or both antagonists, before the addition of 100 nM Ang II for an additional 5 min as indicated. B, COS-7 cells coexpressing the HA-AT2-R and the AT1a-R were pretreated for 10 min with vehicle, 500 nM SP, or 10 µM BIM as indicated before the addition of 100 nM Ang II for an additional 5 min. C, COS-7 cells expressing the indicated receptors were exposed to vehicle or 100 nM Ang II for 5 min as indicated. Solubilized membrane lysates were normalized to an equal number of receptors before immunoprecipitation with the anti-HA antibody. Az, photoaffinity-labeled HA-AT2-Rs.

	Discussion

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These studies have demonstrated that the AT₂-R undergoes PKC-dependent phosphorylation at Ser³⁵⁴ in its cytoplasmic tail during agonist stimulation by Ang II. It is also phosphorylated at the same site during heterologous ligand-mediated PKC activation via the G_q-coupled AT_1a-R. Many GPCRs are coupled through G_q-mediated phosphoinositide hydrolysis to the generation of inositol 1,4,5-trisphosphate, which elevates intracellular [Ca²⁺] and diacylglycerol (DG), which act in conjunction with phosphatidylserine to activate PKC. However, because AT₂-R activation does not lead to inositol 1,4,5-trisphosphate production or Ca²⁺ elevation (Mukoyama et al., 1993), this pathway cannot be responsible for the PKC-dependent phosphorylation observed during agonist stimulation of AT₂-R-expressing COS-7 cells. In the absence of AT₂-R-induced calcium signaling, the inhibitory effect of BIM on AT₂-R phosphorylation suggests that the Ca²⁺-independent or novel isoforms (PKC and ) are involved in this process.

An alternative source of DG production could be AT₂-R-mediated hydrolysis of phosphatidylcholine by phospholipases other than PLC. Because DG is also the source of arachidonic acid, and the AT₂-R has been reported to mediate Ang II-stimulated arachidonic acid release in certain cell types (Lokuta et al., 1994), it is possible that activation of the AT₂-R leads to DG production (possibly from phosphatidylcholine), with resulting release of eicosanoids. This may in turn lead to the activation of a Ca²⁺-independent PKC isoform. In this regard, Ang II stimulates AT₂-R-mediated intracellular alkalinization in cardiac myocytes by a mechanism that is independent of phosphoinositide signaling, but appears to depend on arachidonic acid formation and activation of PKC (Kohout and Rogers, 1995). The AT₂-R has also been reported to mediate Ang II-induced activation and translocation of PKC from cytosol to membrane in cardiac myocytes (Rabkin, 1996).

Agonist-induced receptor phosphorylation via GRKs and/or second messenger-activated kinases has been implicated in the desensitization and internalization mechanisms of many GPCRs (Bohm et al., 1997; Krupnick and Benovic, 1998; Pitcher et al., 1998). The angiotensin AT₁ receptor is phosphorylated at a serine/threonine-rich region of its cytoplasmic tail during agonist activation, and this appears to be mediated largely by GRKs and to a lesser extent by PKC (Smith et al., 1998b; Thomas et al., 1998). Phosphorylation of the AT₁ receptor has been implicated in desensitization and endocytosis of the AT₁ receptor, features that are not associated with activation of the AT₂ receptor.

The functional role of PKC-mediated phosphorylation in AT₂-R action remains to be determined, but does not involve receptor endocytosis because the AT₂-R does not undergo agonist-induced internalization (Hein et al., 1997; Hunyady, 1999). It is possible that phosphorylation of the AT₂-R participates in the initiation or desensitization of its signaling pathways, which are not yet well defined. The ability of the AT₂-R to stimulate SHP-1 tyrosine phosphatase activity (Nouet and Nahmias, 2000) might provide the basis for an assay to detect desensitization of AT₂-R-mediated signaling, comparable to the indices for desensitization of G_s- and G_q-coupled GPCRs provided by measurements of cyclic AMP and inositol phosphate, respectively (Freedman et al., 1995). By analogy with the role that protein kinase A-mediated phosphorylation has been reported to play in switching the G protein coupling of ₂-ARs from G_s to G_i (Daaka et al., 1997), it is also possible that agonist-induced, PKC-mediated phosphorylation of the AT₂-R could influence its G protein coupling specificity. Furthermore, variations in the cell-specific expression of PKC isoforms might explain the apparent differences in G protein coupling observed for AT₂-Rs in different tissues.

The existence of Ang II-mediated homologous and heterologous phosphorylation of the AT₂-R could have significant implications for the understanding of AT₂-R function. Several studies have shown that activation of the AT₂-R counteracts the growth effects of Ang II mediated by the AT₁-R, suggesting that the AT₂-R could provide a brake for the AT₁-dependent hormonal signal. However, little is known about possible converse mechanism(s) for short-term attenuation of the AT₂-R intracellular signal by AT₁-R. It is possible that the interaction between AT₁ and AT₂ receptors has a regulatory role in the physiological actions of Ang II. In this context, the ability of the AT₁-R to transphosphorylate the AT₂-R via PKC in cells expressing both Ang II receptor subtypes reveals a potential mechanism for the modulation of AT₂ receptor-mediated counteraction of AT₁ receptor function. We have recently observed that activation of the endogenous histamine receptor in COS-7 cells also stimulates phosphorylation of the AT₂-R (J. Olivares-Reyes and R. Smith, unpublished data), suggesting that this mechanism could also apply to other G_q-coupled receptors.