Jak2 inhibits GHR endocytosis.

<p>A. GHR-expressing Hek293 cells were transfected with empty vector (EV), Flag-Jak2, Flag-Jak2(Y119E) or Flag-Jak2(1-525). One coverslip was incubated for 15 min with Cy3-GH at 37°C, fixed and stained with anti-Flag (left panels). For EV- and Jak2-transfected cells, cells were also incubated with Cy3-GH and Alexa488-transferrin (Tf) for 15 min at 37°C (right panel). Bar, 5 µm. B. Hek293 GHR or GHR(UbE mutant)-expressing cells were transfected with Jak2 DNA or empty vector and, after 2 days, incubated for 2h on ice with 180 ng/ml ¹²⁵I-GH. To measure uptake kinetics unbound label was removed and the cells were incubated at 37°C for 10 min. C. Hek293 cells were transfected as in 1A and treated with GH for 15 min at 37°C as indicated. Total cell lysates (TCL, upper panel) and GHR immunoprecipitations (lower panel) were analysed on western blot (WB) using anti-Flag, or anti-phosphotyrosine (pY). 130, mature GHR; 110, precursor GHR. Data are representative of three independent experiments. D. Diagram showing the different binding motifs in the dimerised GHR; Jak2 binds Box1, TrCP binds UbE). The transmembrane domains are the dimerisation domains.