Characterization of a novel anti-peptide antibody that recognizes a specific conformation of the platelet-derived growth factor receptor.

Abstract

Two site-specific anti-peptide antibodies (AbPj and AbP2) were raised against the platelet-derived growth factor (PDGF) receptor. These two sites correspond to amino acid residues 977 through 988 (peptide 1) and 932 through 947 (peptide 2) of the murine PDGF receptor. Both antibodies recognized human and murine PDGF receptors in immunoprecipitation and immunoblotting analyses. None of the antibodies was directed to phosphotyrosine. One of the antibodies (AbP2) showed unusual antigen recognition specificity. This antibody specifically recognized the tyrosine-phosphorylated PDGF receptor and not the unphosphorylated native receptor, suggesting that recognition by this antibody requires a specific conformation that is induced by PDGF-stimulated autophosphorylation.The human platelet-derived growth factor (PDGF) consists of a family of glycoproteins (Mr, 28,000 to 35,000) composed of two homologous but nonidentical polypeptides (A and B) of Mr 14,000 to 18,000 linked by disulfide bonds (1,7,11,17); the B chain of PDGF is a product of c-sis proto-oncogene (8, 23). PDGF initiates cellular replication and transformation by binding to a specific high-affinity cell surface receptor, a transmembrane glycoprotein of 180 kilodaltons (kDa). The PDGF receptor has recently been purified from various sources (3,6,19). The purified receptor displays PDGF-stimulated tyrosine kinase activity, suggesting that tyrosine kinase is an integral part of the receptor molecule (3,19). This conclusion has recently been confirmed by cloning of cDNA for the murine PDGF receptor; the deduced sequence has considerable homology with known tyrosine kinases (24).Unlike the well-studied receptors for epidermal growth factor and insulin, the PDGF receptor is relatively uncharacterized. Studies on biosynthesis and processing of this receptor have recently been aided by the availability of anti-PDGF-receptor antibodies (4, 10, 13). In this communication, we report the generation and characterization of a conformation-specific novel anti-PDGF-receptor antibody. It recognizes a peptide epitope (residues 932 through 947 of murine PDGF receptor) that is normally cryptic but becomes accessible upon receptor phosphorylation. This antibody, unlike other reported reagents, may be useful in future structure-function analyses of the receptor. ed by the chloramine T procedure, and '25I-PDGF was purified as described previously (3). Platelet-poor plasma, which lacks PDGF, was prepared as described previously (21).[_y-32P]ATP. Labeled ATP was prepared with 32pi (ICN) and a -y-prep A kit (Promega Biotec, Madison, Wis.) according to the manufacturers' directions. The [_y-32P]ATP was diluted with unlabeled ATP to a final specific activity of 40 to 60 Ci/mmol. Peptide synthesis. The peptides Glu-Gly-Tyr-Lys-LysLys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg (P2) and Tyr-Thr-Ala-Val-Gln-Pro-Asn-Glu-Ser-Asp-Asn-Asp (Pl), corresponding to the residues 932 through 947 and 977 through 988, respectively, of the murine PDGF-receptor sequence (24), were synthesized with an ...

Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C.

“…Coincident with phosphorylation, PDGF receptor conformation is altered, evidenced by increased accessibility of a cytoplasmic domain epitope for recognition by PDGF receptor peptide antibodies (15,16). One consequence of PDGF receptor self-phosphorylation may be acquisition of a conformation competent to bind and affect function of proteins involved in second messenger generation.…”

Characterization of a Novel Epidermal‐Growth‐Factor‐Receptor‐Related 200‐kDa Tyrosine Kinase in Tumor Cells

“…gp200 was initially detected by its interaction with an antipeptide IgG, AbP2, directed to a 16-amino-acid peptide epitope in the cytoplasmic domain of the /I-type PDGF receptor. We and others [6, 171 have shown earlier that AbP2 is a novel conformation-specific antibody that recognizes only the tyrosine-phosphorylated PDGF receptor and not the unphosphorylated native receptor; however, it is not directed to phosphotyrosine [6]. In vitro mutagenesis studies have revealed that the P2 epitope is probably involved in mitogenic message transmission [ 191. gp200 neither binds to PDGF nor does it interact with any other anti-peptide IgG to the a-type or 6-type PDGF receptor.…”

“…This protein has structural features of a growth-factor receptor, including tyrosine kinase activity, and is recognized by a conformation-specific anti-peptide IgG to the ptype PDGF receptor of 180kDa. This peptide epitope in the unphosphorylated PDGF receptor is cryptic and phosphorylation uncovers this site [6]. Studies conducted with other PDGF-receptor-specific antibodies indicated that gp200 is not an aberrant form of the PDGF receptor.…”

“…This was carried out as described previously except that the electrophoretically separated proteins were transferred to membranes poly(viny1idene difluoride) membranes (Millipore Corp.) by electrophoresis at 4 "C overnight at 0.1 mA followed by 1.5 h at 0.2 mA [6].…”

“…All these antibodies recognize human and murine PDGF receptors in immunoprecipitation and Western blotting [6-81. These antibodies were generated in rabbits using HPLC-purified peptides, according to the method described previously [6]. Monoclonal antibody (425), raised against human A43 1 carcinoma cells, was developed as described [13].…”