Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody.
The platelet-derived growth factor (PDGF) receptor is a 180-kDa membrane glycoprotein. A protein of identical size, lectin affinity, and isoelectric point has been identified as a major substrate for PDGF-activated tyrosine kinase in stimulated 3T3 cells. We have purified this tyrosine-phosphorylated protein 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-D-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 greater than 90% homogeneous by silver stain and by [35S]methionine protein autoradiography have specific high affinity 125I-labeled PDGF binding sites (equilibrium dissociation constant, 0.1 X 10(-9) M). Binding activity enrichment in this preparation reflects an 11,000-fold purification of binding activity in intact cells. These data demonstrate that the 180-kDa substrate of the PDGF-stimulated tyrosine kinase is the PDGF receptor. Furthermore, these methods provide a means of purifying this and other tyrosine kinase substrates from growth factor-stimulated cells.
Daniel
T O
TO
Tremble
P M
PM
Frackelton
A R
AR
Williams
L T
LT
D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D008566:Membranes / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000276:immunology / Q000302:isolation & purification*; D017479:Receptors, Platelet-Derived Growth Factor; D012995:Solubility; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology
D000906:Antibodies; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D019000:Phosphotyrosine; D014443:Tyrosine; D017479:Receptors, Platelet-Derived Growth Factor