Ligand stimulation of PDGF β-receptors leads to autophosphorylation of the regulatory tyrosine 857 and of tyrosine residues that in their phosphorylated form serve as docking sites for Src homology 2 domain-containing proteins. Regulation of the PDGF β-receptor by protein-tyrosine phosphatases is poorly understood. We have investigated PDGF β-receptor dephosphorylation by receptor-like protein-tyrosine phosphatase DEP-1 using a cell line with inducible DEP-1 expression and by characterizing in vitrodephosphorylation of the PDGF β-receptor and of receptor-derived phosphopeptides by DEP-1. After DEP-1 induction PDGF β-receptor·DEP-1 complexes and reduced receptor tyrosine phosphorylation were observed. Phosphopeptide analysis of the PDGF β-receptors from DEP-1-expressing cells and of the receptors dephosphorylated in vitro by DEP-1 demonstrated that dephosphorylation of autophosphorylation sites of the receptor differed and revealed that the regulatory Tyr(P)857 was not a preferred site for DEP-1 dephosphorylation. When dephosphorylation of synthetic receptor-derived peptides was analyzed, the selectivity was reproduced, indicating that amino acid sequence surrounding the phosphorylation sites is the major determinant of selectivity. This notion is supported by the observation that the poorly dephosphorylated Tyr(P)562 and Tyr(P)857, in contrast to other analyzed phosphorylation sites, are surrounded by basic amino acid residues at positions −4 and +3 relative to the tyrosine residue. Our study demonstrates that DEP-1 dephosphorylation of the PDGF β-receptor is site-selective and may lead to modulation, rather than general attenuation, of signaling.

An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (G(s alpha)) (Liebmann, C., Graness, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nurnberg, B., and Wetzker, R. (1996) Biochem. J. 318, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of G(s alpha). G(s alpha) was specifically immunoprecipitated from A431 cells using the anti-alpha(s) antiserum AS 348. Tyrosine phosphorylation of G(s alpha) was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [P-32]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of G(s alpha) in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by Bg, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5'-O-(3-[S-35]thiotriphosphate) binding to G(s alpha). In contrast, the BK-evoked, G(q)-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of G(s alpha) is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. me propose that G(s alpha) may represent a key regulatory protein in the cross talk between the signal transduction pathways of BK and EGF in A431 cells.