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.
Liu
Jun
J
Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65212, USA.
Liao
Zhongji
Z
Camden
Jean
J
Griffin
Korey D
KD
Garrad
Richard C
RC
Santiago-Pérez
Laura I
LI
González
Fernando A
FA
Seye
Cheikh I
CI
Weisman
Gary A
GA
Erb
Laurie
L
D000595:Amino Acid Sequence; D000818:Animals; D001254:Astrocytoma; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D066246:ErbB Receptors / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051418:Focal Adhesion Kinase 2; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014162:Transfection; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology; D018909:src Homology Domains; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C412373:AG 1879; C546385:P2RY2 protein, human; C546387:P2ry2 protein, rat; D010446:Peptide Fragments; D011743:Pyrimidines; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium; D014544:Uridine Triphosphate