GH-induced and constitutive GHR endocytosis share the same characteristics.

<p>A. γ2A_GHR_Jak2 cells (left panel) and γ2A_GHR cells (right panel) were stimulated with 500 ng/ml GH for 90 min at 37°C as indicated. Total cell lysates (TCL) (lower panels) and GHR immunoprecipitates (upper panels) were analysed on western blot with the indicated antibodies. B. γ2A_GHR_Jak2 cells were transfected with control siRNA (lanes 1 and 2), clathrin heavy chain siRNA (CHC) (lanes 3 and 4) or TrCP siRNA (lane 5 and 6). Cells were stimulated with 500 ng/ml GH for 90 min at 37°C as indicated, after which total cell lysates (TCL) (3 lower panels) and GHR immunoprecipitates (upper panel) were analysed on western blot with the indicated antibodies. C. γ2A_Jak2 cells, stably transfected with wild-type GHR (WT), GHR mutated in its DSGxxS motif (DSG), GHR mutated in its UbE motif (UbE) and GHR with all cytosolic tyrosine mutated into phenylalanine residues (Y-less) were stimulated with 500 ng/ml GH at 37°C as indicated, after which total cell lysates (TCL) (2 lower panels) and GHR immunoprecipitates (upper panel) were analysed on western blot with the indicated antibodies. Cell lines used in C are stably transfected with the indicated GHR constructs, resulting in clonal variation of Jak2 levels. Data in A and B are representatives of three independent experiments. Data in C are representatives of two independent experiments. D. Quantification of the GH-induced GHR degradation (Y-less from experiment in C, and the data from the experiment in B), expressed as percentage of mature GHR.