Figure 2.

Phe327 is required for degradation of the truncated GHR(349). (A) GHR(349) or GHR(349)(F327A) expressing ts20 cells were incubated for 2 h on ice with 8 nM ¹²⁵I-GH. Unbound label was removed and the cells were incubated at 30°C for the indicated times. The amounts of internalized ¹²⁵I-GH are plotted as a percentage of the cell-associated radioactivity after binding on ice. Each point in the graph represents the mean value of two experiments performed in duplicate ± SD. ●, GHR(349); □, GHR(349)(F327A). (B) GHR(349) or GHR(349)(F327A) expressing ts20 cells were incubated for 6 min at 30°C with 8 nM ¹²⁵I-GH. The ligand was removed and the cells were incubated for the indicated times at 30°C. At each time point, the amount of cell surface, internalized, and degraded ligand was determined as explained in MATERIALS AND METHODS. The amount of ¹²⁵I-GH is plotted as a percentage of total radioactivity. Each point in the graph represents the mean value of two experiments performed in duplicate ± SD. (Left) Intracellular ¹²⁵I-GH. (Right) Degraded ¹²⁵I-GH in the medium. ●, GHR(349); □, GHR(349)(F327A). (C) GHR(349) or GHR(349)(F327A) expressing ts20 cells were labeled with [³⁵S]methionine for 20 min at 30°C and chased in the absence of radioactivity for the indicated times. GHR was immunoprecipitated with anti-GHR antibody (anti-T). p, precursor GHR (50 kDa); m, mature GHR (70 kDa).