Figure 6

Glypican-1 in lipid rafts coimmunoprecipitates with the activated form of Met and regulates hepatocyte growth factor binding to low- and high-affinity cell surface binding sites. (A) C2C12 myoblasts were transfected with rat glypican-1 (F-Gly) or the non-lipid-raft chimeric glypican-1 (F-GlySyn), as described in the Figure 5 legend. F-Gly and F-GlySyn contained a FLAG epitope at the amino terminus. Forty-eight hours after transfection, the cells were serum-starved for 4 hours and then treated with or without 20 ng/ml [¹²⁵I]HGF for 5 minutes. The cell extracts were incubated with anti-FLAG M2 Affinity Gel for 3 hours at 4°C, and, after several washes, the beads were incubated with heparitinase and chondroitinase ABC for 8 hours. The immunoprecipitated (IPP) bound material was eluted with protein loading buffer and analyzed by Western immunoblotting for total hepatocyte growth factor (HGF) receptor (Met), phospho-Met and glypican-1. The membranes were exposed to a phosphorimager to detect [¹²⁵I]HGF. (B) C2C12 and C6 myoblasts were serum-starved for 4 hours and then treated with or without 10 ng/ml [¹²⁵I]HGF for 2 hours at 4°C. After several washes in ice-cold binding buffer, [¹²⁵I]HGF was eluted with high salt and acid to determine low- and high-affinity binding sites, respectively. Counts per minute (cpm) were determined by γ counting and corrected for protein content in cell extracts. Statistical significance was assessed by two-way analysis of variance and a Bonferroni multiple comparisons posttest. **P < 0.01.