Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma.
We have discovered a soluble form of the platelet-derived growth factor (PDGF) alpha receptor, designated sPDGF-R alpha, that is produced by and secreted into the conditioned medium of the human osteosarcoma cell line, MG-63. Additionally, sPDGF-R alpha activity has been detected in normal human blood plasma and serum. We have achieved partial purification of this protein by column chromatography using three different affinity matrices: anti-PDGF-R alpha monoclonal antibody (mAb) 292.15-Sepharose, PDGF-BB-Sepharose, and wheat germ agglutinin-agarose. All three matrices have been shown to purify a 90-kDa protein that is recognized by mAbs specific for the PDGF-R alpha extracellular domain. sPDGF-R alpha is capable of binding PDGF ligand in solution and can compete with cell-associated PDGF receptors for ligand binding. We provide three pieces of data suggesting that the sPDGF-R alpha is generated by proteolytic clipping of the full-length PDGF-R alpha protein. First, the conditioned medium of an expression cell line transfected with a cDNA construct designed to produce only full-length PDGF-R alpha exhibits sPDGF-R alpha activity. Second, a truncated intracellular fragment of the PDGF-R alpha, presumably representing the intracellular counterpart of the clipped sPDGF-R alpha, can be immunoprecipitated from the MG-63 osteosarcoma cell extracts using antiserum raised against an intracellular portion of PDGF-R alpha. Finally, we have been unable to detect alternative splicing in the PDGF-R alpha transcript using reverse transcription-polymerase chain reaction.
Tiesman
J
J
ZymoGenetics, Inc., Seattle, Washington 98105.
Hart
C E
CE
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D017077:Culture Media, Conditioned; D006801:Humans; D008970:Molecular Weight; D012516:Osteosarcoma; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing; D011863:Radioimmunoassay; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012867:Skin; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
D000906:Antibodies; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D017479:Receptors, Platelet-Derived Growth Factor