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The platelet derived growth factor (PDGF) family comprises of isoforms A-D, which form disulfide-linked homo- and hetero-dimers: PDGF-AA, -BB, -AB, -CC and -DD. These proteins signal through the trans-membrane receptor tyrosine kinases PDGFR-α and PDGFR-β. Binding of two surface receptors to a PDGF-dimer, results in receptor dimerization and autophosphorylation of the intracellular tyrosine kinase domains. These phosphorylated domains provide binding sites for substrates, which are in turn phosphorylated to initiate an intracellular signaling cascade, leading to cell proliferation. PDGFR-α recognizes PDGF A, B and C while PDGFR-β only recognizes B and D. Therefore, homodimers of PDGFR-α bind PDGF-AA, -AB, -BB or -CC, while PDGFR-β homodimers bind PDGF-BB and -DD. PDGFR heterodimers (α/β) can be activated by PDGF-AB, -BB and -CC. As well as being involved in angiogenesis, PDGF signaling regulates the growth of fibroblasts, smooth muscle cells and glial cells. Aberrant PDGF signaling is implicated in fibrotic conditions, atherosclerosis and human malignancies, including melanomas, gliomas and prostate cancer. Mouse fibroblast A31 cells express PDGFR-α and PDGFR-β. A capture ELISA has been developed to measure PDGF-BB stimulated PDGFR auto-phosphorylation in A31 cell lysates, enabling the evaluation of drug effects on the kinase activity of the receptor. The assay format was optimized to allow for growth, treatment and lysis of cells in a 96-well tissue culture plate, followed by the transfer of the lysates to ELISA plates precoated with a rabbit polyclonal antibody (Upstate 06-495) specific for both PDGFR-α and PDGFR-β. The phosphorylation of the captured PDGFR protein was quantified using a phosphotyrosine specific Ab (PY20), directly labeled with alkaline phosphatase (AP) and a chemiluminescent AP-substrate. From a library of kinase inhibitors, the inhibition of PDGFR autophosphorylation was highly correlated with their anti-proliferative activity. The selectivity of protein kinase inhibitors is usually assessed through their effects on the phosphorylation of artificial substrates catalyzed by the recombinant kinase domains of these enzymes. Such inhibitory profiles can significantly differ to profiles obtained with full-length enzymes in cells. STI571 (Imatinib; Gleevec™) is an established inhibitor of the Bcr-Abl, c-Kit and PDGFR kinases. In cellular assays STI571 inhibits PDGFR, c-Kit and Bcr-Abl with IC50 values of 73 ± 12 nM (n = 10; A31 cells), 90 ± 10 nM (n = 7; GIST882 cells; Mestan et al. Blood 2003) and 191 ± 10 nM (n = 7; 32-D Bcr-Abl cells), whereas it inhibits the catalytic activity of recombinant PDGFR, Kit and Abl with IC50 values of 869 ± 117 nM (n = 12; [ATP] 1.0 μM), 579 ± 92 nM (n = 11; [ATP] 1.0 μM) and 170 ± 23 nM (n = 21; [ATP] 5.0 μM). Thus the kinase selectivity profile of STI571 markedly differs in cellular assays compared to that obtained using recombinant kinases.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]