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 40-45 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-2197 cells. The addition to U-2197 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 Cl 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 alpha-receptor.
Afrakhte
M
M
Department of Pathology, University Hospital, Uppsala, Sweden.
Nistér
M
M
Ostman
A
A
Westermark
B
B
Paulsson
Y
Y
D000368:Aged; D017398:Alternative Splicing; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000737:chemistry / Q000378:metabolism; D018219:Histiocytoma, Benign Fibrous / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011233:Precipitin Tests / Q000379:methods; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000032:analysis; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D010982:Platelet-Derived Growth Factor; D012334:RNA, Neoplasm; C075278:platelet-derived growth factor A; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor