Angiotensin II-induced Association of Phospholipase Cgamma 1 with the G-protein-coupled AT1 Receptor*

Richard C. VenemaDagger , Hong JuDagger , Virginia J. VenemaDagger , Bernhard Schieffer§, Joyce B. Harp, Brian N. Lingpar , Douglas C. Eatonpar , and Mario B. Marreropar **

From the Dagger  Vascular Biology Center, Department of Pediatrics, and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, the § Division of Cardiology, Hannover Medical School, Hannover, Germany, the  Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina 27599, and the par  Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

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 Cgamma 1 (PLCgamma 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 PLCgamma 1 to the AT1 receptor in an Ang II- and tyrosine phophorylation-dependent manner. The PLCgamma 1-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 PLCgamma 1. PLCgamma 1 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

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 Cgamma (PLCgamma ), 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 C-terminal 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 PLCgamma 1 to the platelet-derived growth factor alpha  and beta 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 PLCgamma 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-protein-coupled receptors that contain seven membrane-spanning alpha -helices. These receptors lack intrinsic tyrosine kinase activity. However, we have previously shown that the G-protein-coupled angiotensin II (Ang II) AT1 receptor in vascular smooth muscle cells (VSMC) activates the inositol 1,4,5-trisphosphate (IP3) and diacylglycerol-generating enzyme, PLCgamma 1, in a manner similar to that observed for growth factor receptors. PLCgamma 1 is transiently tyrosine-phosphorylated in Ang II-stimulated VSMC with a time course that parallels that of IP3 formation (5). Tyrosine phosphorylation of PLCgamma 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 PLCgamma 1 and blocks Ang II stimulation of IP3 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 PLCgamma 1 (7-9). In none of these instances, however, is it known whether PLCgamma 1 phosphorylation and activation involves physical association of the SH2 domains of the enzyme with the receptor.

AT1 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 AT1 receptor activates the JAK/STAT pathway by inducing rapid 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 AT1 receptor that is identical to the PLCgamma 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 AT1 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 PLCgamma 1 in VSMC involves binding of PLCgamma 1 to the AT1 receptor in an Ang II- and tyrosine phosphorylation-dependent manner. In addition, we have identified the interacting domains in the two proteins.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Anti-AT1 receptor polyclonal antibodies (N-10 and 306) and glutathione S-transferase (GST)-PLCgamma 1 fusion proteins (sc-4019, sc-4051, sc-4052, sc-4053, and sc-4054) were purchased from Santa Cruz Biotechnology Inc. Anti-PLCgamma 1 monoclonal antibody (clone D-7-3) and anti-phosphotyrosine monoclonal antibody (clone 4G10) were obtained from Upstate Biotechnology. Anti-SHP-2 monoclonal antibody was purchased from Transduction Laboratories. Purified human recombinant c-Src enzyme and PP1 came from Calbiochem. Affi-Gel 10, Muta-Gene Phagemid kit, and detergent-compatible protein assay kit were purchased from Bio-Rad. [3H]PIP2, GST-agarose, and monoclonal anti-GST antibody were obtained from Amersham Pharmacia Biotech. All other chemicals were purchased from Sigma.

Cell Culture-- VSMC from 200-300 g male Sprague-Dawley rat aortas were cultured to near confluence at 37 °C under 5% CO2 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 (10-7 M) for various times, and cells were lysed and subjected to immunoprecipitation with anti-AT1 receptor antibody as described previously (10). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose by electroblotting, and probed with anti-PLCgamma 1 or anti-phosphotyrosine antibody as described previously (5, 6).

Preparation of VSMC Cell Lysates-- Growth-arrested VSMC were stimulated with Ang II (10-7 M) for various times, washed two times with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 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 Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin). Cells were scraped off the plates and gently sonicated. Lysates were cleared by centrifugation at 7,500 × 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-AT1 Receptor Fusion Proteins-- A 166-base pair fragment of the Ca18b cDNA encoding the rat AT1A 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-AT1 fusion proteins were expressed in DH5alpha Escherichia coli and purified by affinity chromatography using immobilized glutathione-Sepharose 4B beads. Five µg 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 nitrocellulose by electroblotting, and immunoblotted with anti-PLCgamma 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 AT1 receptor to PLCgamma 1, GST fusion proteins containing the various SH2 and SH3 domains of PLCgamma 1 were utilized.

Assay of PLC Activity-- PLC activity was assayed using [3H]PIP2-containing liposomes as substrate as described previously by Goldschmidt-Clermont et al. (14).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

To determine whether PLCgamma 1 associates with the AT1 receptor in a ligand- and tyrosine phosphorylation-dependent manner, we utilized a rabbit polyclonal anti-AT1 receptor antibody directed against the C-terminal 54 amino acid residues (306-359) of the rat AT1A receptor (12). Cultured VSMC were stimulated with Ang II (10-7 M) for various times, cells were lysed, and the AT1 receptor was immunoprecipitated from the lysates with anti-AT1 receptor antibody. Immunoprecipitated proteins were separated by gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-PLCgamma 1 antibody. As shown in Fig. 1, Ang II induced a rapid and transient association of PLCgamma 1 (140 kDa) with the AT1 receptor that was maximal within 30 s to 1 min. The time course of Ang II-stimulated PLCgamma 1-AT1 receptor association is thus similar to that reported previously for Ang II-stimulated PLCgamma 1 tyrosine phosphorylation and activation(5). Identical results were also obtained when the experiments were repeated using a different rabbit polyclonal anti-AT1 receptor antibody that recognizes residues 15-24 in the N terminus of the rat AT1A receptor (data not shown). However, in negative control experiments using rabbit preimmune serum or an irrelevant rabbit polyclonal anti-GST antibody, no PLCgamma 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 PLCgamma 1 with the AT1 receptor, we also carried out coimmunoprecipitation experiments in which cells were pretreated with the Src family kinase-selective inhibitor, PP1 (10-6 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 AT1 receptor-PLCgamma 1 association (data not shown), suggesting that tyrosine phosphorylation of either PLCgamma 1 or the receptor (or both) by a Src family tyrosine kinase may be required for the association. This possibility appears plausible because both PLCgamma 1 and the AT1 receptor have been shown previously to be excellent substrates for Src kinases in vitro (13, 16). Furthermore, PLCgamma 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 PLCgamma 1 or AT1 receptor phosphorylation in a tyrosine phosphorylation cascade.


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Fig. 1.   Time course of Ang II-stimulated association of PLCgamma 1 with the AT1 receptor in VSMC. VSMC were stimulated for the times shown with Ang II (10-7 M). Cells were lysed, and cleared supernatants were immunoprecipitated with anti-AT1 receptor antibody. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-PLCgamma 1 antibody. Shown is a single blot (inset) and densitometric analysis of blots from four separate experiments (mean ± S.E.). In the three blots not shown, significant binding of PLCgamma 1 to the receptor was detected at 3 min.

To confirm the results of the coimmunoprecipitation experiments and to determine whether PLCgamma 1 binds to the AT1 receptor C-terminal intracellular domain, we utilized a GST-AT1 fusion protein (GST-AT1-(306-359)) containing the C-terminal 54 amino acids of the rat AT1A receptor. The GST-AT1 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 (10-7 M) for various times, and cell lysates were prepared and used in in vitro binding assays with the GST-AT1-(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 PLCgamma 1 eluted (and therefore bound by the fusion protein) was then quantitated by immunoblotting with anti-PLCgamma 1 antibody. As shown in Fig. 2, lysates from Ang II-treated VSMC induced the binding of PLCgamma 1 to the GST-AT1-(306-359) fusion protein with a time course that was similar to that observed for PLCgamma 1 binding to the AT1 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 (10-6 M for 30 min) before Ang II stimulation, PLCgamma 1 binding to the fusion protein was completely blocked (data not shown).


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Fig. 2.   Time course of Ang II-stimulated association of PLCgamma 1 in VSMC lysates with the GST-AT1-(306-359) fusion protein. VSMC were stimulated with Ang II (10-7 M) for the times indicated and then lysed. Lysates were used in in vitro binding assays with the GST-AT1-(306-359) fusion protein. Lysates were incubated with the fusion protein prebound to agarose beads for 2 h at 4 °C. Incubation of the fusion protein with Ang II-treated lysates resulted in tyrosine phosphorylation of the protein, as confirmed by immunoblotting with anti-phosphotyrosine. Beads were washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. Binding of PLCgamma 1 to the fusion protein was quantitated by immunoblotting with anti-PLCgamma 1 antibody. Shown is a representative blot (inset) and densitometric analysis of blots from four separate experiments (mean ± S.E.).

Loss of binding of PLCgamma 1 to the GST-AT1-(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 AT1 receptor C-terminal intracellular domain. This phosphorylation event may, in turn, be required for PLCgamma 1 binding to the receptor. To determine whether the GST-AT1 fusion protein becomes tyrosine phosphorylated when incubated with lysates from Ang II-treated VSMC, cells were either treated or not treated with Ang II (10-7 M for 30 s), and lysates were prepared and then incubated with the GST-AT1-(306-359) fusion protein prebound to agarose beads. Experiments were also carried out in which cells were pretreated with PP1 (10-6 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 anti-phosphotyrosine antibody. As shown in Fig. 3, lysates from Ang II-treated VSMC induced the tyrosine phosphorylation of the GST-AT1 fusion protein. Ang II-stimulated phosphorylation, however, was completely blocked when cells were pretreated with PP1. Furthermore, phosphorylation was restricted to the AT1 receptor portion of the fusion protein, as no tyrosine phosphorylation of the GST alone negative control was detected (data not shown).


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Fig. 3.   Tyrosine phosphorylation of the GST-AT1-(306-359) fusion protein by Ang II-treated VSMC lysates. VSMC were either treated or not treated with Ang II (10-7 M for 30 s) following either pretreatment or no pretreatment with PP1 (10-6 M for 30 min). Cells were lysed and incubated with the GST-AT1-(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.

Recently we have shown that JAK2 association with the AT1 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-AT1-(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-AT1-(306-359) fusion protein.2 In the present study, we have tested whether quantitative depletion of SHP-2 from VSMC lysates also alters PLCgamma 1 binding to the GST-AT1-(306-359) fusion protein. Lysates were prepared from Ang II-treated (10-7 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-AT1-(306-359) fusion protein prebound to beads. PLCgamma 1 binding to the fusion protein and to GST alone in the two conditions was quantitated by immunoblotting with anti-PLCgamma 1 antibody as before. As shown in Fig. 4, PLCgamma 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 PLCgamma 1 association with the AT1 receptor, unlike that of the JAK2 tyrosine kinase, does not depend on SHP-2 acting as an adaptor protein for PLCgamma 1 binding.


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Fig. 4.   Effect of immunodepletion of SHP-2 on binding of PLCgamma 1 in Ang II-treated VSMC lysates to the GST-AT1-(306-359) fusion protein. VSMC were treated with Ang II (10-7 M for 30 s), cells were lysed, and lysates were either immunoprecipitated or not immunoprecipitated (control) with anti-SHP-2 antibody. Quantitative removal of SHP-2 from lysates by immunoprecipitation was confirmed by immunoblotting. Immunodepleted and nonimmunodepleted lysates were then used in in vitro assays with the GST-AT1-(306-359) fusion protein or GST alone prebound to agarose beads. After a 2-h incubation at 4 °C, beads were washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. Binding of PLCgamma 1 to the fusion protein was quantitated by immunoblotting with anti-PLCgamma 1 antibody. Similar results were obtained in two separate experiments.

The hypothesis that c-Src or other Src family tyrosine kinase modulates the PLCgamma 1-AT1 receptor association is supported further by the results of binding competition experiments with a GST-AT1-(306-359) fusion protein phosphorylated in vitro by c-Src. In these experiments, the GST-AT1-(306-359) fusion protein was first covalently linked to an agarose matrix and then allowed to bind PLCgamma 1 in VSMC lysates prepared from cells exposed to Ang II (10-7 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 anti-phosphotyrosine antibody. Free nonphosphorylated and phosphorylated forms of the GST-AT1-(306-359) fusion protein were then used to compete with the immobilized GST fusion protein for binding of PLCgamma 1. The amount of PLCgamma 1 remaining bound to the immobilized fusion protein after incubation with the competitor proteins was quantitated by immunoblotting of glutathione-eluted proteins with anti-PLCgamma 1 antibody. As shown in Fig. 5, no competition was observed with the nonphosphorylated protein. However, increasing concentrations of free tyrosine-phosphorylated GST-AT1 fusion protein effectively competed with the GST-AT1 receptor fusion protein agarose matrix for PLCgamma 1 binding, suggesting that direct phosphorylation of the AT1 receptor C-terminal tail by c-Src may increase its binding affinity for PLCgamma 1.


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Fig. 5.   Competition of free phosphorylated and nonphosphorylated GST-AT1-(306-359) fusion proteins with an immobilized GST-AT1-(306-359) fusion protein for binding by PLCgamma 1. VSMC were treated with Ang II (10-7 M for 30 s), and cell lysates were prepared. Proteins in lysates were allowed to bind to the GST-AT1-(306-359) fusion protein immobilized on Affi-Gel 10. Nonphosphorylated and c-Src-phosphorylated free GST-AT1-(306-359) fusion proteins at various concentrations were then incubated for 30 min at 4 °C with the immobilized GST fusion protein prebound by PLCgamma 1. The amount of PLCgamma 1 that remained bound to the immobilized GST-AT1-(306-359) was quantitated by immunoblotting with anti-PLCgamma 1 antibody. Similar results were obtained in three separate experiments.

Inhibition of PLCgamma 1 binding to the immobilized GST-AT1 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 PLCgamma 1 binding to the AT1 receptor, we carried out in vitro binding assays with GST-AT1-(306-359) fusion proteins that were either phosphorylated or not phosphorylated in vitro by c-Src. The AT1 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 PLCgamma 1, we individually mutated each tyrosine residue in the GST-AT1-(306-359) fusion protein to a phenylalanine. Wild-type GST-AT1-(306-359), GST-AT1-(306-359) ( Tyr-312 right-arrow Phe), GST-AT1-(306-359) (Tyr-319 right-arrow Phe), and GST-AT1-(306-359) (Tyr-339 right-arrow 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 PLCgamma 1 from untreated VSMC lysates. Binding was quantitated by immunoblotting with anti-PLCgamma 1 antibody as described earlier. As shown in Fig. 6, PLCgamma 1 in untreated lysates bound to the wild-type GST-AT1 receptor fusion protein only if it had been phosphorylated in vitro by c-Src. PLCgamma 1 also bound to in vitro phosphorylated GST-AT1-(306-359) (Tyr-312 right-arrow Phe) and GST-AT1-(306-359) (Tyr-339 right-arrow Phe) fusion proteins but not to the GST-AT1-(306-359) (Tyr-319 right-arrow Phe) fusion protein, demonstrating that it is phosphorylation of Tyr-319 specifically that is required for tyrosine phosphorylation-dependent association of PLCgamma 1 with the AT1 receptor.


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Fig. 6.   Effect of phosphorylation of GST-AT1 fusion proteins by c-Src in vitro on binding of the fusion proteins by PLCgamma 1. The wild-type GST-AT1-(306-359) fusion protein prebound to agarose beads was either phosphorylated or not phosphorylated in vitro by purified recombinant human c-Src. GST-AT1 fusion proteins containing mutated tyrosines (at 312, 319, and 339) were also phosphorylated in vitro by c-Src. Nonphosphorylated and phosphorylated proteins prebound to beads were washed extensively with buffer containing M NaCl before use in in vitro binding assays with lysates from untreated VSMC. After a 2-h incubation at 4 °C, beads were again washed extensively with buffer containing 1 M NaCl, and bound proteins were eluted. Binding of PLCgamma 1 to the fusion proteins was quantitated by immunoblotting with anti-PLCgamma 1 antibody. Similar results were obtained in two separate experiments. Y, Tyr; F, Phe.

In order for tyrosine phosphorylation of the AT1 receptor C-terminal intracellular domain to have a role in mediating PLCgamma 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 AT1 receptor in VSMC, untreated cells or cells treated with Ang II (10-7 M for 30 s) were lysed and immunoprecipitated with anti-AT1 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 (10-4 M for 30 min), or PP1 (10-6 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 AT1 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.


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Fig. 7.   Ang II-stimulated tyrosine phosphorylation of the AT1 receptor in VSMC. VSMC were either treated or not treated with Ang II (10-7 M for 30 s) following either pretreatment or no pretreatment (control) with sodium orthovanadate (10-4 M for 30 min) or PP1 (10-6 for 30 min). Cells were lysed and immunoprecipitated with anti-AT1 receptor antibody, and immunoprecipitated proteins were immunoblotted with anti-phosphotyrosine antibody. In the experiment shown, two differentially glycosylated forms of the AT1 receptor were not resolved. Similar results were obtained in two separate experiments.

To further map the region of the AT1 receptor C-terminal tail that interacts with PLCgamma 1, we expressed a series to GST-AT1 fusion proteins containing various deletional or point mutations in the AT1 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 PLCgamma 1 in lysates from Ang II-treated (10-7 M for 30 s) VSMC. Binding was detected by immunoblotting with anti-PLCgamma 1 as described earlier. Fusion proteins of the AT1 receptor containing residues 306-359, 306-348, 306-329, and 318-359 were each bound by PLCgamma 1. In contrast, fusion proteins containing AT1 receptor residues 336-359, 323-359, and 306-318 were not bound by PLCgamma 1 (Fig. 8A). Deletional analysis thus identifies residues located between positions 318 and 323 as being essential for PLCgamma 1 binding. The YIPP motif in the AT1 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 PLCgamma 1 and that, as shown also in Fig. 6, phosphorylation of tyrosine 319 within the motif enhances PLCgamma 1 binding in a manner similar to that shown previously for the platelet-derived growth factor alpha  and beta  receptors. This conclusion is also supported by the results of in vitro binding assays using VSMC lysates from Ang II-treated cells (10-7 M for 30 s) and the GST-AT1-(306-359) fusion proteins in which the tyrosine residues at positions 312, 319, and 339 of the AT1 receptor were each individually mutated to phenylalanines. Assays were carried out with Ang II-treated (10-7 M for 30 s) VSMC lysates (which as shown earlier contain activated Src family tyrosine kinases that can phosphorylate the fusion proteins) and either wild-type GST-AT1-(306-359), GST-AT1-(306-359) (Tyr-312 right-arrow Phe), GST-AT1-(306-359) (Tyr-319 right-arrow Phe), or GST-AT1-(306-359) (Tyr-339 right-arrow Phe) fusion proteins. As shown in Fig. 8B, PLCgamma 1 from VSMC lysates bound to each of the fusion proteins with the exception of GST-AT1-(306-359) (Tyr-319 right-arrow Phe), again indicating an essential role for tyrosine 319 in PLCgamma 1 binding.

                              
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Table I
GST-AT1 receptor fusion proteins


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Fig. 8.   Mutational analysis of AT1 receptor amino acids required for in vitro binding of PLCgamma 1 to GST-AT1 fusion proteins. VSMC were treated with Ang II (10-7 M for 30 s) and cell lysates were prepared and used in in vitro binding assays with GST-AT1 fusion proteins containing various deletional or point mutations in the AT1 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 PLCgamma 1 eluted (and therefore bound by a given fusion protein) was quantitated by immunoblotting with anti-PLCgamma 1 antibody. Similar results were obtained in three separate experiments. Y, Tyr; F, Phe.

The importance of tyrosine 319 in PLCgamma 1 binding to the AT1 receptor and in activation of the PLCgamma 1 enzyme was further confirmed in in vitro binding assays in which PLCgamma 1 binding to the receptor was quantitated by PLC activity. Ang II-treated VSMC lysates (10-7 M for 30 s) were incubated with wild-type GST-AT1-(306-359), GST-AT1-(329-359), GST-AT1-(306-359) (Tyr-319 right-arrow Phe), GST-AT1-(306-359) (Tyr-312 right-arrow Phe), and GST-AT1-(306-359) (Tyr-339 right-arrow 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 [3H]PIP2-containing liposomes as substrate. As shown in Fig. 9, a deletional mutant (323-359) lacking the YIPP motif and the point mutant (Tyr-319 right-arrow Phe) lacking tyrosine 319 bound very little PLC activity, whereas other mutants lacking tyrosines 312 (Tyr-312 right-arrow Phe) and 339 (Tyr-339 right-arrow Phe) bound significantly more PLC activity, equivalent to that bound by the wild-type fusion protein.


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Fig. 9.   Mutational analysis of AT1 receptor amino acids required for association of PLC activity with GST-AT1 fusion proteins. VSMC were treated with Ang II (10-7 M for 30 s), and cell lysates were prepared and mixed with the indicated GST-AT1 fusion proteins prebound to agarose beads. After extensive washing of the beads, PLC activity was determined using [3H]PIP2-containing liposomes as substrate. Results shown represent mean ± S.E. from three separate experiments. Y, Tyr; F, Phe.

Full-length PLCgamma 1 contains two SH2 domains and a single SH3 domain. To determine which of these domains, if any, are required for interaction of PLCgamma 1 with the AT1 receptor, we also carried out in vitro binding assays with commercially available GST fusion proteins containing the various SH2 and SH3 domains of PLCgamma 1 (Fig. 10A). VSMC were exposed to Ang II (10-7 M) for 0, 0.5, and 1 min and then lysed. Lysates were incubated with GST-PLCgamma 1 fusion proteins prebound to agarose beads. Beads were washed extensively, and bound proteins were eluted with reduced glutathione. The amount of AT1 receptor eluted was then quantitated by immunoblotting with anti-AT1 receptor antibody. As shown in Fig. 10B, only the C-terminal SH2 domain of PLCgamma 1 (residues 663-760 of the rat PLCgamma 1 sequence) was required for ligand-dependent binding of the enzyme to the AT1 receptor.


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Fig. 10.   Deletional analysis of Src homology domains of PLCgamma 1 required for binding to the AT1 receptor. VSMC were treated with Ang II (10-7 M) for 0, 0.5, and 1 min and then lysed. Lysates were incubated with the various GST-PLCgamma 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 AT1 receptor eluted was quantitated by immunoblotting with anti-AT1 receptor antibody. In the experiment shown, two differentially glycosylated forms of the AT1 receptor were resolved. Similar results were obtained in three separate experiments.

In summary, the results of the present study show for the first time that PLCgamma 1 binds to the G-protein-coupled AT1 receptor in an Ang II- and tyrosine phosphorylation-dependent manner. The PLCgamma 1-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 SH2 domains in PLCgamma 1. PLCgamma 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.

    ACKNOWLEDGEMENT

We wish to thank Dr. Kenneth E. Bernstein in whose laboratory the GST-AT1 receptor cDNA constructs were originally prepared.

    FOOTNOTES

* 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. The 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{at}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; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]; JAK, janus kinase; STAT, signal transducers and activators of transcription; GST, glutathione-S-transferase.

2 V. J. Venema, M. B. Marrero, H. Ju, B. Li, J. Li, D. C. Eaton, and R. C. Venema, submitted for publication.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

  1. Fantl, W. J., Johnson, D. E., and Williams, L. T. (1993) Annu. Rev. Biochem. 62, 453-481[CrossRef][Medline] [Order article via Infotrieve]
  2. Malarkey, K., Belham, C. M., Paul, A., Graham, A., McLees, A., Scott, P. H., Plevin, R. (1995) Biochem. J. 309, 361-375[Medline] [Order article via Infotrieve]
  3. Valius, M., Bazenet, C., and Kazlauskas, A. (1993) Mol. Cell. Biol. 13, 133-143[Abstract]
  4. Eriksson, A., Nånberg, E., Rönnstrand, L., Engström, U., Hellman, U., Rupp, E., Carpenter, G., Heldin, C-H., and Claesson-Welsh, L. (1995) J. Biol. Chem. 270, 7773-7781[Abstract/Free Full Text]
  5. Marrero, M. B., Paxton, W. G., Duff, J. L., Berk, B. C., Bernstein, K. E. (1994) J. Biol. Chem. 269, 10935-10939[Abstract/Free Full Text]
  6. Marrero, M. B., Schieffer, B., Paxton, W. G., Schieffer, E., Bernstein, K. E. (1995) J. Biol. Chem. 270, 15734-15738[Abstract/Free Full Text]
  7. Dhar, A., and Shukla, S. D. (1994) J. Biol. Chem. 269, 9123-9127[Abstract/Free Full Text]
  8. Rao, G. N., Delafontaine, P., and Runge, M. S. (1995) J. Biol. Chem. 270, 27871-27875[Abstract/Free Full Text]
  9. Puceat, M., and Vassort, G. (1996) Biochem. J. 318, 723-728[Medline] [Order article via Infotrieve]
  10. Marrero, M. B., Schieffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., Bernstein, K. E. (1995) Nature 375, 247-250[CrossRef][Medline] [Order article via Infotrieve]
  11. Ali, M. S., Syeski, P. P., Dirksen, L. B., Hayzer, D. J., Marrero, M. B., Bernstein, K. E. (1997) J. Biol. Chem. 272, 23382-23388[Abstract/Free Full Text]
  12. Murphy, T. J., Alexander, R. W., Griendling, K. K., Runge, M. S., Bernstein, K. E. (1991) Nature 351, 233-236[CrossRef][Medline] [Order article via Infotrieve]
  13. Paxton, W. G., Marrero, M. B., Klein, J. D., Delafontaine, P., Berk, B. C., Bernstein, K. E. (1994) Biochem. Biophys. Res. Commun. 200, 260-267[CrossRef][Medline] [Order article via Infotrieve]
  14. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., Pollard, T. D. (1991) Science 251, 1231-1233[Medline] [Order article via Infotrieve]
  15. Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H., Weringer, E. J., Pollok, B. A., Connelly, P. A. (1996) J. Biol. Chem. 271, 695-701[Abstract/Free Full Text]
  16. Liao, F., Shin, H. S., and Rhee, S. G. (1993) Biochem. Biophys. Res. Commun. 191, 1028-1033[CrossRef][Medline] [Order article via Infotrieve]
  17. Nakanishi, O., Shibasaki, F., Hidaka, M., Homma, Y., and Takenawa, T. (1993) J. Biol. Chem. 268, 10754-10759[Abstract/Free Full Text]
  18. Khare, S., Bolt, M. J. G., Wali, R. K., Skarosi, S. F., Roy, H. K., Niedziela, S., Scaglione-Sewell, B., Aquino, B., Sitrin, M. D., Brasitus, T. A., Bissonnette, M. (1997) J. Clin. Invest. 99, 1831-1841[Abstract/Free Full Text]


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