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.
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 P2Y(2) nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated ... with the P2Y(2) receptor in 1321N1 astrocytoma cells stimulated with the P2Y(2) receptor agonist UTP. A mutant P2Y(2) 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 P2Y(2) 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 P2Y(2) receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y(2) receptor and allows Src to efficiently phosphorylate the EGFR.
Mesh Terms:
Amino Acid Sequence, Animals, Astrocytoma, Binding Sites, Calcium, Cell Membrane, Fluorescent Antibody Technique, Focal Adhesion Kinase 2, Humans, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Molecular Sequence Data, Mutagenesis, PC12 Cells, Peptide Fragments, Phosphorylation, Protein-Tyrosine Kinases, Pyrimidines, Rats, Receptor, Epidermal Growth Factor, Receptors, Platelet-Derived Growth Factor, Receptors, Purinergic P2, Receptors, Purinergic P2Y2, Transfection, Tumor Cells, Cultured, Uridine Triphosphate, src Homology Domains, src-Family Kinases
Amino Acid Sequence, Animals, Astrocytoma, Binding Sites, Calcium, Cell Membrane, Fluorescent Antibody Technique, Focal Adhesion Kinase 2, Humans, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Molecular Sequence Data, Mutagenesis, PC12 Cells, Peptide Fragments, Phosphorylation, Protein-Tyrosine Kinases, Pyrimidines, Rats, Receptor, Epidermal Growth Factor, Receptors, Platelet-Derived Growth Factor, Receptors, Purinergic P2, Receptors, Purinergic P2Y2, Transfection, Tumor Cells, Cultured, Uridine Triphosphate, src Homology Domains, src-Family Kinases
J. Biol. Chem.
Date: Feb. 27, 2004
PubMed ID: 14670955
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