Demonstration of Functionally Different Interactions between Phospholipase C-γ and the Two Types of Platelet-derived Growth Factor Receptors

Abstract

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…As shown in Figure 2b and c, a phosphorylated Tyr-762 peptide (pY762) dose-dependently disrupted complex formation between the PDGF a-receptor and CrkII or CrkL, whereas the unphosphorylated control peptide (Y762) showed no e ect. Another phosphorylated peptide, pY988, which contains another autophosphorylation site in the areceptor (Eriksson et al, 1995;Yokote et al, 1996), did not either show any e ect. The results suggest that Crk proteins speci®cally recognize phosphorylated Tyr-762 in the PDGF a-receptor in vivo.…”

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site

“…This selectivity is consistent with the residues carboxy-terminal of Ufo Y821 (pYVNM). The PLCg domain speci®city also seems to include amino acids in position +4, +5 and +6, as well as residues N-terminal relative to phosphotyrosine (Pascal et al, 1994;Larose et al, 1993Larose et al, , 1995Eriksson et al, 1995). A comparison between the amino acid sequences surrounding the phosphorylated tyrosine residues involved in binding of PLCg to the Ufo receptor, and various other receptors, i.e.…”

SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis

“…After incubation with 50 mM Na 3 VO 4 for 30 min at 378C, the¯asks were transferred to ice, and stimulated or not with 100 ng/ml PDGF-BB, for 1 h on ice. Cells were washed twice with ice-cold PBS, lysed and processed for either immunoprecipitation with a-SHP-2 antibodies (Santa Cruz) or incubation with wheat germ agglutinin-Sepharose, as described (Eriksson et al, 1995). After boiling in reducing Laemmli bu er, samples were separated by SDS-gel electrophoresis on a 7% gel, electrotransferred to an Immobilon P membrane, blocked with bovine serum albumin (BSA) in PBS overnight, and probed with PY20 antiphosphotyrosine antibodies, essentially as described by Blume-Jensen et al (1993).…”