Production of cell‐associated PDGF‐AA by a human sarcoma cell line: evidence for a latent autocrine effect

The alternative splicing of platelet‐derived growth factor A‐chain is known to result in 2 different protein products. One variant is encoded by transcripts containing the 69 nts representing exon 6 (PDGF‐AA L ), and one variant is encoded by transcripts in which exon 6 is excluded (PDGF‐AA s ). Transfection assays have suggested that the long splice variant of the A‐chain is mainly associated with membrane‐ and matrix‐associated heparan sulphate proteoglycans, whereas the shorter variant is soluble. We describe a human sarcoma cell line (U‐2197) that expresses a high level of PDGF‐A transcripts. Immunoprecipitations revealed cell‐associated protein products of mainly 24, 28 and 33 kDa and less abundant forms of 4—45 kDa, while no PDGF was found in the medium. Analysis of extracellular medium in a radioreceptor assay confirmed that PDGF was not secreted by the U‐2 197 cells. The addition to U‐2 197 cultures of a carboxy terminal peptide that specifically competes with the binding of the long splice variant of PDGF‐AA to extracellular matrix and cell membranes resulted in the release of 3 PDGF‐AA‐specific dimeric proteins with molecular masses of 33, 37 and 45 kDa. Furthermore, polymerase chain reaction studies discriminating between the long and the short splice variants of the PDGF‐A transcripts revealed that U‐2197 expressed relatively higher amounts of the long splice variant compared with U‐343 MGa C1 2:6, which is known to secrete PDGF‐AA. These cell‐associated forms of PDGF, released to the medium by adding carboxy terminal peptide, increased the tyrosine kinase activity of the endogenous PDGF α‐receptor. © 1996 Wiley‐Liss, Inc.