Angiotensin II-induced association of phospholipase Cgamma1 with the G-protein-coupled AT1 receptor.

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase Cgamma1 (PLCgamma1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLCgamma1 to the AT1 receptor in an Ang II- and tyrosine phophorylation-dependent manner. The PLCgamma1-AT1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLCgamma1. PLCgamma1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase.JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT 1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase C␥1 (PLC␥1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLC␥1 to the AT 1 receptor in an Ang II-and tyrosine phophorylation-dependent manner. The PLC␥1-AT 1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT 1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLC␥1. PLC␥1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase⅐JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT 1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.
Growth factor receptors belong to a family of receptors that contain an extracellular ligand binding domain, a single transmembrane portion, and a large intracellular tyrosine kinase catalytic domain. Ligand-induced receptor autophosphorylation promotes the interaction of the intracellular domains of the receptors with a number of downstream effector proteins or enzymes. Typically, these proteins contain one or more domains known as Src homology 2 (SH2) 1 domains. Among these SH2 domain-containing proteins are phosphoinositide-specific phospholipase C␥ (PLC␥), the 85-kDa subunit of phosphatidylinositol 3-kinase, GTPase-activating proteins, growth factor receptor binding protein 2, the phosphotyrosine phosphatase SHP-2, and members of the nonreceptor Src family of tyrosine kinases (1,2). Autophosphorylation of growth factor receptors occurs on defined tyrosine residues. These phosphorylated residues function to initiate cellular signaling cascades by acting as high affinity binding sites for the SH2 domains of various effector proteins. The selectivity of the receptor-effector interaction is determined, not only by the phosphorylated tyrosine residue in the receptor but also by the three amino acids Cterminal to the phosphorylated tyrosine and by the structure of the SH2 domain of the interacting protein. For example, one of the identified sites for binding of the SH2 domains of PLC␥1 to the platelet-derived growth factor ␣ and ␤ receptors is a YIPP motif present in the receptors at residues 1018 -1021 and 1021-1024, respectively. Phosphorylation of tyrosines 1018 and 1021 in these motifs promotes binding of PLC␥1 to the platelet-derived growth factor receptor and tyrosine phosphorylation and activation of the enzyme (3,4).
Another family of cell surface receptors are the G-proteincoupled receptors that contain seven membrane-spanning ␣-helices. These receptors lack intrinsic tyrosine kinase activity. However, we have previously shown that the G-proteincoupled angiotensin II (Ang II) AT 1 receptor in vascular smooth muscle cells (VSMC) activates the inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol-generating enzyme, PLC␥1, in a manner similar to that observed for growth factor receptors. PLC␥1 is transiently tyrosine-phosphorylated in Ang II-stimulated VSMC with a time course that parallels that of IP 3 formation (5). Tyrosine phosphorylation of PLC␥1 appears to lie downstream from activation of the c-Src tyrosine kinase because electroporation of neutralizing anti-c-Src antibodies into VSMC virtually eliminates Ang II-induced tyrosine phosphorylation of PLC␥1 and blocks Ang II stimulation of IP 3 production (6). Furthermore, other G-protein-coupled receptors, including those for platelet activating factor, thrombin, and ATP, have also been shown to signal through the tyrosine phosphorylation and activation of PLC␥1 (7)(8)(9). In none of these instances, however, is it known whether PLC␥1 phosphorylation and activation involves physical association of the SH2 domains of the enzyme with the receptor.
AT 1 post-receptor signaling in VSMC also involves activation of the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Ang II stimulation of the AT 1 receptor activates the JAK/STAT pathway by inducing rapid * This work was supported by National Institutes of Health Grants P01-DK50268, DK-02111, HL57201, and HL58139, a Veterans Affairs Merit Review Award, an American Diabetes Association Research Award, an American Heart Association grant-in-aid award, an American Heart Association/Astra-Merck grant-in-aid award, and an American Heart Association Minority Scientist Developmental Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Emory University School of Medicine, The Center for Cell and Molecular Signaling, Physiology Bldg., 1648 Pierce Dr. N.E., Atlanta, GA 30322. Tel.: 404-727-3310; Fax: 404-727-0329; E-mail: mmarrero@ccms-renal. physio.emory.edu. 1 The abbreviations used are: SH2, Src homology 2; PLC, phospholipase C; Ang II, angiotensin II; VSMC, vascular smooth muscle cells; IP 3 , inositol 1,4,5-trisphosphate; PIP 2 , phosphatidylinositol 4,5bisphosphate; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-tyrosine phosphorylation, activation, and association of JAK2 with the receptor (10). JAK2-receptor association appears to depend on a YIPP motif in the C-terminal intracellular domain of the AT 1 receptor that is identical to the PLC␥1 SH2 domain binding site identified in the platelet-derived growth factor receptor (11). Because JAK2 does not contain any SH2 domains, the finding that JAK2 associates with this motif in the AT 1 receptor was initially puzzling. Recently, however, we have found that JAK2 associates with the receptor as a consequence of the SH2 domain-containing SHP-2 phosphotyrosine phosphatase acting as an adaptor or linker protein for JAK2 association. 2 In the present study, we have examined whether Ang II-induced tyrosine phosphorylation and activation of PLC␥1 in VSMC involves binding of PLC␥1 to the AT 1 receptor in an Ang II-and tyrosine phosphorylation-dependent manner. In addition, we have identified the interacting domains in the two proteins.
Cell Culture-VSMC from 200 -300 g male Sprague-Dawley rat aortas were cultured to near confluence at 37°C under 5% CO 2 in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and supplemented with antibiotics (5,6). Cells were growth-arrested by incubation in serum-free Dulbecco's modified Eagle medium for 36 -48 h before Ang II exposure.
Immunoprecipitation and Immunoblotting-VSMC were stimulated with Ang II (10 Ϫ7 M) for various times, and cells were lysed and subjected to immunoprecipitation with anti-AT 1 receptor antibody as described previously (10). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose by electroblotting, and probed with anti-PLC␥1 or anti-phosphotyrosine antibody as described previously (5,6).
Preparation of VSMC Cell Lysates-Growth-arrested VSMC were stimulated with Ang II (10 Ϫ7 M) for various times, washed two times with ice-cold phosphate-buffered saline containing 1 mM Na 3 VO 4 and then lysed in 1.0 ml of lysis buffer (25 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 1% Triton X-100, 10% glycerol, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin). Cells were scraped off the plates and gently sonicated. Lysates were cleared by centrifugation at 7,500 ϫ g for 15 min, and the protein concentration of the cleared lysates was determined by the Bio-Rad detergent-compatible protein assay. In some experiments, SHP-2 was quantitatively removed (as confirmed by immunoblotting) from VSMC lysates by immunoprecipitation with anti-SHP-2 antibody before use of the lysates in in vitro binding assays.
Preparation of DNA Constructs Encoding GST-AT 1 Receptor Fusion Proteins-A 166-base pair fragment of the Ca18b cDNA encoding the rat AT 1A receptor was amplified by the polymerase chain reaction and cloned into the pGEX-KG vector via XbaI and HindIII restriction sites (12). Point mutations and deletional mutations were introduced into the constructs as described previously (11). The sequences of all DNA constructs were verified by DNA sequence analysis.
In Vitro Binding Assays-GST-AT 1 fusion proteins were expressed in DH5␣ Escherichia coli and purified by affinity chromatography using immobilized glutathione-Sepharose 4B beads. Five g of fusion protein or GST alone prebound to beads was incubated with 1.0 ml of VSMC cell lysate (0.9 -1.0 mg of protein) for 2 h at 4°C. The beads were then washed four times with ice-cold lysis buffer containing 1 M NaCl. Bound proteins were eluted by boiling in SDS sample buffer. Eluted proteins were separated on 7.5% SDS-polyacrylamide gels, transferred to nitro-cellulose by electroblotting, and immunoblotted with anti-PLC␥1 antibody. In some experiments GST fusion proteins were covalently linked to Affi-Gel 10 according to the manufacturer's instructions for use in binding competition experiments. In other experiments, GST fusion proteins were phosphorylated in vitro by c-Src as described previously (13), before use of the fusion proteins in in vitro binding assays. For studies of in vitro binding of the full-length AT 1 receptor to PLC␥1, GST fusion proteins containing the various SH2 and SH3 domains of PLC␥1 were utilized.
Assay of PLC Activity-PLC activity was assayed using [ 3 H]PIP 2containing liposomes as substrate as described previously by Goldschmidt-Clermont et al. (14).

RESULTS AND DISCUSSION
To determine whether PLC␥1 associates with the AT 1 receptor in a ligand-and tyrosine phosphorylation-dependent manner, we utilized a rabbit polyclonal anti-AT 1 receptor antibody directed against the C-terminal 54 amino acid residues (306 -359) of the rat AT 1A receptor (12). Cultured VSMC were stimulated with Ang II (10 Ϫ7 M) for various times, cells were lysed, and the AT 1 receptor was immunoprecipitated from the lysates with anti-AT 1 receptor antibody. Immunoprecipitated proteins were separated by gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-PLC␥1 antibody. As shown in Fig. 1, Ang II induced a rapid and transient association of PLC␥1 (140 kDa) with the AT 1 receptor that was maximal within 30 s to 1 min. The time course of Ang II-stimulated PLC␥1-AT 1 receptor association is thus similar to that reported previously for Ang II-stimulated PLC␥1 tyrosine phosphorylation and activation (5). Identical results were also obtained when the experiments were repeated using a different rabbit polyclonal anti-AT 1 receptor antibody that recognizes residues 15-24 in the N terminus of the rat AT 1A receptor (data not shown). However, in negative control experiments using rabbit preimmune serum or an irrelevant rabbit polyclonal anti-GST antibody, no PLC␥1 was immunoprecipitated for any of the time points. To investigate whether phosphorylation by an Src family tyrosine kinase is required for the Ang II-induced association of PLC␥1 with the AT 1 receptor, we also carried out coimmunoprecipitation experiments in which cells were pretreated with the Src family kinase-selective inhibitor, PP1 (10 Ϫ6 M for 30 min) before Ang II stimulation. PP1, which has been shown previously to be highly selective for Src family kinases relative to other known tyrosine kinases (15), completely prevented AT 1 receptor-PLC␥1 association (data not shown), suggesting that tyrosine phosphorylation of either FIG. 1. Time course of Ang II-stimulated association of PLC␥1 with the AT 1 receptor in VSMC. VSMC were stimulated for the times shown with Ang II (10 Ϫ7 M). Cells were lysed, and cleared supernatants were immunoprecipitated with anti-AT 1 receptor antibody. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-PLC␥1 antibody. Shown is a single blot (inset) and densitometric analysis of blots from four separate experiments (mean Ϯ S.E.). In the three blots not shown, significant binding of PLC␥1 to the receptor was detected at 3 min. PLC␥1 or the receptor (or both) by a Src family tyrosine kinase may be required for the association. This possibility appears plausible because both PLC␥1 and the AT 1 receptor have been shown previously to be excellent substrates for Src kinases in vitro (13,16). Furthermore, PLC␥1 has been shown to form a complex with c-Src in several other cell types (7,17,18). An alternative explanation for the inhibitory effect of PP1 is that Src kinase activity may be required for a phosphorylation event that is upstream from either PLC␥1 or AT 1 receptor phosphorylation in a tyrosine phosphorylation cascade.
To confirm the results of the coimmunoprecipitation experiments and to determine whether PLC␥1 binds to the AT 1 receptor C-terminal intracellular domain, we utilized a GST-AT 1 fusion protein (GST-AT 1 -(306 -359)) containing the C-terminal 54 amino acids of the rat AT 1A receptor. The GST-AT 1 fusion and GST alone were expressed in E. coli and purified to homogeneity on a glutathione-agarose affinity column. VSMC were treated with Ang II (10 Ϫ7 M) for various times, and cell lysates were prepared and used in in vitro binding assays with the GST-AT 1 -(306 -359) fusion protein prebound to agarose beads. In control experiments, lysates were also incubated with GST alone prebound to agarose beads. After a 2-h incubation at 4°C, the beads were washed extensively in buffer containing 1 M NaCl, and bound proteins were eluted. The amount of PLC␥1 eluted (and therefore bound by the fusion protein) was then quantitated by immunoblotting with anti-PLC␥1 antibody. As shown in Fig. 2, lysates from Ang II-treated VSMC induced the binding of PLC␥1 to the GST-AT 1 -(306 -359) fusion protein with a time course that was similar to that observed for PLC␥1 binding to the AT 1 receptor in intact cells. No binding was detected for the GST alone negative control (data not shown). Furthermore, when the cells from which lysates were prepared were pretreated with PP1 (10 Ϫ6 M for 30 min) before Ang II stimulation, PLC␥1 binding to the fusion protein was completely blocked (data not shown).
Loss of binding of PLC␥1 to the GST-AT 1 -(306 -359) fusion protein as a consequence of PP1 pretreatment suggests that binding requires the activity of c-Src or other Src family tyrosine kinases. One possibility is that Src kinase activity in the VSMC lysates is required for direct tyrosine phosphorylation of the AT 1 receptor C-terminal intracellular domain. This phos-phorylation event may, in turn, be required for PLC␥1 binding to the receptor. To determine whether the GST-AT 1 fusion protein becomes tyrosine phosphorylated when incubated with lysates from Ang II-treated VSMC, cells were either treated or not treated with Ang II (10 Ϫ7 M for 30 s), and lysates were prepared and then incubated with the GST-AT 1 -(306 -359) fusion protein prebound to agarose beads. Experiments were also carried out in which cells were pretreated with PP1 (10 Ϫ6 M for 30 min) prior to Ang II stimulation. After a 2-h incubation at 4°C, the beads were washed extensively, and bound proteins were eluted. The relative phosphotyrosine content of the eluted fusion protein was then assessed by immunoblotting with antiphosphotyrosine antibody. As shown in Fig. 3, lysates from Ang II-treated VSMC induced the tyrosine phosphorylation of the GST-AT 1 fusion protein. Ang II-stimulated phosphorylation, however, was completely blocked when cells were pretreated with PP1. Furthermore, phosphorylation was restricted to the AT 1 receptor portion of the fusion protein, as no tyrosine phosphorylation of the GST alone negative control was detected (data not shown).
Recently we have shown that JAK2 association with the AT 1 receptor involves the SH2 domain-containing SHP-2 phosphotyrosine phosphatase acting as an adaptor protein for JAK2 association. This conclusion is based on in vitro binding assays with Ang II-treated VSMC lysates and the GST-AT 1 -(306 -359) fusion protein in which lysates were quantitatively depleted of SHP-2 by immunoprecipitation with anti-SHP-2 antibody before determining the extent of JAK2 binding to the fusion protein. Immunodepletion of lysates with anti-SHP-2 completely blocks JAK2 association with the GST-AT 1 -(306 -359) fusion protein. 2 In the present study, we have tested whether quantitative depletion of SHP-2 from VSMC lysates also alters PLC␥1 binding to the GST-AT 1 -(306 -359) fusion protein. Lysates were prepared from Ang II-treated (10 Ϫ7 M for 30 s) VSMC and then depleted of SHP-2 by immunoprecipitation with anti-SHP-2 antibody. Quantitative depletion of SHP-2 from lysates was confirmed by immunoblotting with anti-SHP-2 antibody. Nondepleted (control) lysates were also prepared. Immunodepleted and nondepleted lysates were then used in in vitro binding assays with the GST-AT 1 -(306 -359) fusion protein prebound to beads. PLC␥1 binding to the fusion protein and to GST alone in the two conditions was quantitated by immunoblotting with anti-PLC␥1 antibody as before. As shown in Fig. 4, PLC␥1 bound to the fusion protein to approximately the same extent whether from SHP-2-depleted or nondepleted lysates. No binding was detected for the GST alone negative control. Therefore, we conclude that PLC␥1 association with the AT 1 receptor, unlike that of the JAK2 tyrosine kinase, does not depend on SHP-2 acting as an adaptor protein for PLC␥1 binding.  (306 -359) fusion protein by Ang II-treated VSMC lysates. VSMC were either treated or not treated with Ang II (10 Ϫ7 M for 30 s) following either pretreatment or no pretreatment with PP1 (10 Ϫ6 M for 30 min). Cells were lysed and incubated with the GST-AT 1 -(306 -359) fusion protein prebound to agarose beads for 2 h at 4°C. Beads were then washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. Bound proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose by electroblotting, and immunoblotted with anti-phosphotyrosine. Similar results were obtained in two separate experiments.
The hypothesis that c-Src or other Src family tyrosine kinase modulates the PLC␥1-AT 1 receptor association is supported further by the results of binding competition experiments with a GST-AT 1 -(306 -359) fusion protein phosphorylated in vitro by c-Src. In these experiments, the GST-AT 1 -(306 -359) fusion protein was first covalently linked to an agarose matrix and then allowed to bind PLC␥1 in VSMC lysates prepared from cells exposed to Ang II (10 Ϫ7 M for 30 s). In addition, the purified free GST fusion protein was either treated or not treated with purified human recombinant c-Src and MgATP to obtain phosphorylated and nonphosphorylated forms of the protein. Tyrosine phosphorylation of the fusion protein by c-Src in vitro was confirmed by immunoblotting of anti-GST immunoprecipitates with antiphosphotyrosine antibody. Free nonphosphorylated and phosphorylated forms of the GST-AT 1 -(306 -359) fusion protein were then used to compete with the immobilized GST fusion protein for binding of PLC␥1. The amount of PLC␥1 remaining bound to the immobilized fusion protein after incubation with the competitor proteins was quantitated by immunoblotting of glutathioneeluted proteins with anti-PLC␥1 antibody. As shown in Fig. 5, no competition was observed with the nonphosphorylated protein.
However, increasing concentrations of free tyrosine-phosphorylated GST-AT 1 fusion protein effectively competed with the GST-AT 1 receptor fusion protein agarose matrix for PLC␥1 binding, suggesting that direct phosphorylation of the AT 1 receptor Cterminal tail by c-Src may increase its binding affinity for PLC␥1.
Inhibition of PLC␥1 binding to the immobilized GST-AT 1 receptor fusion protein by the free phosphorylated but not the nonphosphorylated fusion protein could also be due to an indirect, allosteric interference rather than to competition for the binding site. Therefore, to more directly demonstrate a role for receptor phosphorylation in PLC␥1 binding to the AT 1 receptor, we carried out in vitro binding assays with GST-AT 1 -(306 -359) fusion proteins that were either phosphorylated or not phosphorylated in vitro by c-Src. The AT 1 receptor C-terminal cytoplasmic tail (residues 306 -359) contains tyrosine residues at positions 312, 319, and 339. To determine whether phosphorylation of one or more of these residues is required for binding of PLC␥1, we individually mutated each tyrosine residue in the GST-AT 1 -(306 -359) fusion protein to a phenylalanine. Wildtype GST-AT 1 -(306 -359), GST-AT 1 -(306 -359) ( Tyr-312 3 Phe), GST-AT 1 -(306 -359) (Tyr-319 3 Phe), and GST-AT 1 -(306 -359) (Tyr-339 3 Phe) fusion proteins prebound to agarose beads were each treated with purified c-Src and MgATP in vitro to obtain phosphorylated forms of the proteins. Phosphorylated fusion proteins and the nonphosphorylated wild-type fusion protein were then used in in vitro binding assays to detect possible binding by PLC␥1 from untreated VSMC lysates. Binding was quantitated by immunoblotting with anti-PLC␥1 antibody as described earlier. As shown in Fig. 6, PLC␥1 in untreated lysates bound to the wild-type GST-AT 1 receptor fusion protein only if it had been phosphorylated in vitro by c-Src. PLC␥1 also bound to in vitro phosphorylated GST-AT 1 -(306 -359) (Tyr-312 3 Phe) and GST-AT 1 -(306 -359) (Tyr-339 3 Phe) fusion proteins but not to the GST-AT 1 -(306 -359) (Tyr-319 3 Phe) fusion protein, demonstrating that it is phosphorylation of Tyr-319 specifically that is required for tyrosine phosphorylation-dependent association of PLC␥1 with the AT 1 receptor. In order for tyrosine phosphorylation of the AT 1 receptor C-terminal intracellular domain to have a role in mediating PLC␥1 binding to the receptor in VSMC, it must occur rapidly (within 30 s) in response to Ang II stimulation. To investigate whether Ang II induces rapid tyrosine phosphorylation of the AT 1 receptor in VSMC, untreated cells or cells treated with Ang II (10 Ϫ7 M for 30 s) were lysed and immunoprecipitated with anti-AT 1 receptor antibody. Immunoprecipitates were then immunoblotted with anti-phosphotyrosine antibody. Experiments were also carried out in which VSMC were pretreated with either the tyrosine phosphatase inhibitor, sodium orthovanadate (10 Ϫ4 M for 30 min), or PP1 (10 Ϫ6 M for 30 min). Results shown in Fig. 7 demonstrate that Ang II induces a rapid and significant increase in the phosphotyrosine content of the AT 1 receptor in VSMC. Pretreatment with sodium orthovanadate increased the phosphotyrosine content of the receptor even in the absence of Ang II stimulation. In contrast, pretreatment with PP1 completely abolished the Ang II-induced tyrosine phosphorylation of the receptor, suggesting a requirement for Src family kinase activity in receptor phosphorylation.
To further map the region of the AT 1 receptor C-terminal tail that interacts with PLC␥1, we expressed a series to GST-AT 1 fusion proteins containing various deletional or point mutations in the AT 1 portion of the fusion protein. Proteins were expressed in E. coli and purified by affinity chromatography on glutathione-agarose (Table I). Each mutant protein was then individually tested for its ability to bind PLC␥1 in lysates from Ang II-treated (10 Ϫ7 M for 30 s) VSMC. Binding was detected by immunoblotting with anti-PLC␥1 as described earlier. Fusion proteins of the AT 1 receptor containing residues 306 -359, 306 -348, 306 -329, and 318 -359 were each bound by PLC␥1. In contrast, fusion proteins containing AT 1 receptor residues 336 -359, 323-359, and 306 -318 were not bound by PLC␥1 (Fig. 8A). Deletional analysis thus identifies residues located between positions 318 and 323 as being essential for PLC␥1 binding. The YIPP motif in the AT 1 receptor C-terminal tail, which has been shown previously to bind the JAK/SHP-2 complex, is located at positions 318 -322. Thus it is likely that this motif also functions as a binding site for PLC␥1 and that, as shown also in Fig. 6, phosphorylation of tyrosine 319 within the motif enhances PLC␥1 binding in a manner similar to that shown previously for the platelet-derived growth factor ␣ and ␤ receptors. This conclusion is also supported by the results of in vitro binding assays using VSMC lysates from Ang II-treated cells ( The importance of tyrosine 319 in PLC␥1 binding to the AT 1 receptor and in activation of the PLC␥1 enzyme was further confirmed in in vitro binding assays in which PLC␥1 binding to the receptor was quantitated by PLC activity. Ang II-treated VSMC lysates (10 Ϫ7 M for 30 s) were incubated with wild-type GST-AT 1 -(306 -359), GST-AT 1 -(329 -359), GST-AT 1 -(306 -359) (Tyr-319 3 Phe), GST-AT 1 -(306 -359) (Tyr-312 3 Phe), and GST-AT 1 -(306 -359) (Tyr-339 3 Phe) fusion proteins prebound to agarose beads. Beads were washed extensively, and proteins were eluted with reduced glutathione. Eluates were then assayed for PLC activity using [ 3 H]PIP 2 -containing liposomes as substrate. As shown in Fig. 9, a deletional mutant (323-359) lacking the YIPP motif and the point mutant (Tyr-319 3 Phe) lacking tyrosine 319 bound very little PLC activity, whereas other mutants lacking tyrosines 312 (Tyr-312 3 Phe) and 339 (Tyr-339 3 Phe) bound significantly more PLC activity, equivalent to that bound by the wild-type fusion protein.
Full-length PLC␥1 contains two SH2 domains and a single SH3 domain. To determine which of these domains, if any, are required for interaction of PLC␥1 with the AT 1 receptor, we also carried out in vitro binding assays with commercially available GST fusion proteins containing the various SH2 and SH3 domains of PLC␥1 (Fig. 10A). VSMC were exposed to Ang II (10 Ϫ7 M) for 0, 0.5, and 1 min and then lysed. Lysates were incubated with GST-PLC␥1 fusion proteins prebound to agarose beads. Beads were washed extensively, and bound proteins were eluted with reduced glutathione. The amount of AT 1 receptor eluted was then quantitated by immunoblotting with FIG. 7. Ang II-stimulated tyrosine phosphorylation of the AT 1 receptor in VSMC. VSMC were either treated or not treated with Ang II (10 Ϫ7 M for 30 s) following either pretreatment or no pretreatment (control) with sodium orthovanadate (10 Ϫ4 M for 30 min) or PP1 (10 Ϫ6 for 30 min). Cells were lysed and immunoprecipitated with anti-AT 1 receptor antibody, and immunoprecipitated proteins were immunoblotted with anti-phosphotyrosine antibody. In the experiment shown, two differentially glycosylated forms of the AT 1 receptor were not resolved. Similar results were obtained in two separate experiments. anti-AT 1 receptor antibody. As shown in Fig. 10B, only the C-terminal SH2 domain of PLC␥1 (residues 663-760 of the rat PLC␥1 sequence) was required for ligand-dependent binding of the enzyme to the AT 1 receptor.
In summary, the results of the present study show for the first time that PLC␥1 binds to the G-protein-coupled AT 1 receptor in an Ang II-and tyrosine phosphorylation-dependent manner. The PLC␥1-AT 1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT 1 receptor and binding of the phosphorylated receptor by the most C-terminal of two SH2 domains in PLC␥1. PLC␥1 thus binds to the same site in the receptor previously identified for binding of the SHP-2 phosphotyrosine phosphatase/JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. The data presented here further demonstrates that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors. FIG. 8. Mutational analysis of AT 1 receptor amino acids required for in vitro binding of PLC␥1 to GST-AT 1 fusion proteins. VSMC were treated with Ang II (10 Ϫ7 M for 30 s) and cell lysates were prepared and used in in vitro binding assays with GST-AT 1 fusion proteins containing various deletional or point mutations in the AT 1 receptor C-terminal intracellular domain. Lysates were incubated with each of the different fusion proteins (prebound to agarose beads) for 2 h. Beads were then washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. The amount of PLC␥1 eluted (and therefore bound by a given fusion protein) was quantitated by immunoblotting with anti-PLC␥1 antibody. Similar results were obtained in three separate experiments. Y, Tyr; F, Phe.
FIG. 9. Mutational analysis of AT 1 receptor amino acids required for association of PLC activity with GST-AT 1 fusion proteins. VSMC were treated with Ang II (10 Ϫ7 M for 30 s), and cell lysates were prepared and mixed with the indicated GST-AT 1 fusion proteins prebound to agarose beads. After extensive washing of the beads, PLC activity was determined using [ 3 H]PIP 2 -containing liposomes as substrate. Results shown represent mean Ϯ S.E. from three separate experiments. Y, Tyr; F, Phe.
FIG. 10. Deletional analysis of Src homology domains of PLC␥1 required for binding to the AT 1 receptor. VSMC were treated with Ang II (10 Ϫ7 M) for 0, 0.5, and 1 min and then lysed. Lysates were incubated with the various GST-PLC␥1 fusion proteins prebound to agarose beads. Beads were washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. The amount of AT 1 receptor eluted was quantitated by immunoblotting with anti-AT 1 receptor antibody. In the experiment shown, two differentially glycosylated forms of the AT 1 receptor were resolved. Similar results were obtained in three separate experiments.