sphingosine-1-phosphate and monodansylcadaverine

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.

Topics: Adaptor Proteins, Signal Transducing; Animals; Arrestins; beta-Adrenergic Receptor Kinases; beta-Arrestins; Cadaverine; Cell Line; Cells, Cultured; Concanavalin A; Cyclic AMP-Dependent Protein Kinases; Dynamin II; Endocytosis; Enzyme Inhibitors; GRB2 Adaptor Protein; Guinea Pigs; Humans; Immunoprecipitation; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Receptor, Platelet-Derived Growth Factor beta; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Transfection; Transport Vesicles