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

Eewa Nånberg,Eva Rupp,Graham Carpenter,Anders Eriksson,Lars Rönnstrand,Ulla Engström,Ulf Hellman,Carl-Henrik Heldin,Lena Claesson-Welsh

doi:10.1074/jbc.270.13.7773

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.

Highlights

We report the mapping of two autophosphorylation sites, Tyr-988 and Tyr-1018, in the carboxyl-terminal tail ofthe human PDGF a-receptor and show that these sites are required for the interaction of the a-receptor with phospholipase C-y (PLC-y)
To characterize the potential role of the PDGF a-receptor carboxyl-terminal tyrosine residues in receptor signaling, we analyzed whether they serve as autophosphorylation sites
We have identified two autophosphorylation sites, Tyr-988 and Tyr-l018, near the carboxyl terminus of the human PDGF a-receptor, which are phosphorylated both in vivo and in vitro

Summary

Introduction

The phosphorylated tyrosine residues of the receptor form specific binding sites for downstream signaling components, containing one or more -100-amino acid residue motifs denoted Src homology 2 (SH2) domains (reviewed in Ref. 5). Eight autophosphorylation sites have been mapped in the PDGF J3-receptor: Tyr-579 and Tyr-581 in the juxtamembrane region [7], Tyr-740, Tyr-751, and Tyr-771 in the kinase insert [8, 9], Tyr-857 in the kinase domain [10], and Tyr-l009 and Tyr-l021 in the carboxyl-terminal tail [11,12,13,14] Seven of these residues are conserved in the PDGF a-receptor at positions that are homologous with those in the J3-receptor. We show that the human PDGF a- and J3-receptors differ quantitatively in their abilities to associate with and phosphorylate PLC-y and to stimulate inositol phosphate production

Methods

Results

Conclusion