PURPOSE: To biochemically characterize the receptor for connective tissue growth factor (CTGF) of human corneal fibroblasts (HCF). METHODS: Radiolabeled recombinant human CTGF was used to determine the specificity and time course of binding to low-passage cultures of HCF. The affinity and number of receptors present were calculated by Scatchard and best-fit analyses. In vitro immunoprecipitation assays with radiolabeled CTGF and soluble mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF-2-R) alone, or with CTGF-related growth factors were conducted. Additionally, (125)I-CTGF-binding and CTGF-stimulated proliferation were measured in cultures of M6P/IGF-2-R knockout fibroblasts. RESULTS: Binding of (125)I-CTGF to fibroblast cultures was significantly displaced by CTGF, but not by related growth factors. Scatchard plot analysis indicated the presence of both a high-affinity, low-abundance binding site, and a low-affinity, high-abundance binding site; whereas, the best-fit analysis suggests a single high-affinity, low-abundance binding site. A 280 kDa complex containing cross-linked (125)I-CTGF was immunoprecipitated by antibodies to CTGF or M6P/IGF-2-R. M6P/IGF-2-R knockout cells have a reduced proliferative response to TGF-β, and don't proliferate at all in response to CTGF. CONCLUSIONS: CTGF binds to the M6P/IGF-2-R with high affinity, and the M6P/IGF-2-R is required for CTGF-stimulated proliferation in fibroblasts. These observations suggest that the M6P/IGF-2-R may be a new antifibrotic target.
PURPOSE: To biochemically characterize the receptor for connective tissue growth factor (CTGF) of human corneal fibroblasts (HCF). METHODS: Radiolabeled recombinant humanCTGF was used to determine the specificity and time course of binding to low-passage cultures of HCF. The affinity and number of receptors present were calculated by Scatchard and best-fit analyses. In vitro immunoprecipitation assays with radiolabeled CTGF and soluble mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF-2-R) alone, or with CTGF-related growth factors were conducted. Additionally, (125)I-CTGF-binding and CTGF-stimulated proliferation were measured in cultures of M6P/IGF-2-R knockout fibroblasts. RESULTS: Binding of (125)I-CTGF to fibroblast cultures was significantly displaced by CTGF, but not by related growth factors. Scatchard plot analysis indicated the presence of both a high-affinity, low-abundance binding site, and a low-affinity, high-abundance binding site; whereas, the best-fit analysis suggests a single high-affinity, low-abundance binding site. A 280 kDa complex containing cross-linked (125)I-CTGF was immunoprecipitated by antibodies to CTGF or M6P/IGF-2-R. M6P/IGF-2-R knockout cells have a reduced proliferative response to TGF-β, and don't proliferate at all in response to CTGF. CONCLUSIONS:CTGF binds to the M6P/IGF-2-R with high affinity, and the M6P/IGF-2-R is required for CTGF-stimulated proliferation in fibroblasts. These observations suggest that the M6P/IGF-2-R may be a new antifibrotic target.
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