A Connective Tissue Growth Factor Signaling Receptor in Corneal Fibroblasts

Abstract

To biochemically characterize the receptor for connective tissue growth factor (CTGF) of human corneal fibroblasts (HCF). 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. 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. 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.