Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody.

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Abstract
The platelet-derived growth factor (PDGF) receptor is a 180-kDa [kilodalton] membrane glycoprotein. A protein of identical size, lectin affinity and isoelectric point was identified as a major substrate for PDGF-activated tyrosine kinase in stimulated 3T3 cells. This tyrosine-phosphorylated protein was purified to homogeneity by using anti-phosphotyrosine immunoaffinity and lectin affinity steps. Demonstration that this purified tyrosine phosphoprotein is the PDGF receptor necessitated development of an assay capable of identifying specific 125I-labeled PDGF binding activity in soluble receptor preparations. PDGF receptor solubilized from 3T3 cell membranes with the detergent octyl .beta.-L-glucoside was precipitated on an artificial liposome matrix after receptor aggregation with concanavalin A. Precipitated binding sites display affinity and kinetic characteristics of PDGF receptors in cells and membranes. Preparations of the 180-kDa phosphoprotein that are > 90% homogeneous by Ag stain and by [35S]Met protein autoradiography have specific high affinity 125I-labeled PDGF binding sites (equilibrium Kd 0.1 .times. 10-9 M). Binding activity enrichment in this preparation reflects an 11,000-fold purification of binding activity in intact cells. The 180-kDa substrate of the PDGF-stimulated tyrosine kinase is the PDGF receptor. These methods provide a means of purifying this and other tyrosine kinase substrates from growth factor-stimulated cells.

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