Src Homology 3 Binding Sites in the P2Y2 Nucleotide Receptor Interact with Src and Regulate Activities of Src, Proline-rich Tyrosine Kinase 2, and Growth Factor Receptors

• Published in: The Journal of biological chemistry Vol. 279; no. 9; pp. 8212 - 8218
• Main Authors: Liu, Jun, Liao, Zhongji, Camden, Jean, Griffin, Korey D., Garrad, Richard C., Santiago-Pérez, Laura I., González, Fernando A., Seye, Cheikh I., Weisman, Gary A., Erb, Laurie
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27-02-2004; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Astrocytoma; Binding Sites; Calcium - metabolism; Cell Membrane - chemistry; ErbB Receptors - metabolism; Fluorescent Antibody Technique; Focal Adhesion Kinase 2; Humans; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases - metabolism; Molecular Sequence Data; Mutagenesis; PC12 Cells; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Phosphorylation; Protein-Tyrosine Kinases - metabolism; Pyrimidines - pharmacology; Rats; Receptors, Platelet-Derived Growth Factor - metabolism; Receptors, Purinergic P2 - chemistry; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2Y2; src Homology Domains; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - metabolism; Transfection; Tumor Cells, Cultured; Uridine Triphosphate - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M312230200

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y2 nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y2 receptor in 1321N1 astrocytoma cells stimulated with the P2Y2 receptor agonist UTP. A mutant P2Y2 receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y2 receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y2 receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y2 receptor and allows Src to efficiently phosphorylate the EGFR.