Figure 4: The interaction of Gαs with the GPCR endosomal-sorting machinery is independent of the activation status of Gαs.
From: Gαs regulates the post-endocytic sorting of G protein-coupled receptors
(a) In vitro GST pull-down experiment showing that the Gαs activation state did not influence its direct interactions with HRS, dysbindin and GASP-1. In vitro translated 35S-labelled HRS, dysbindin and GASP-1 were incubated with GST alone or GST–Gαs preloaded with GDP (to mimic the inactive state) or GDP/AlF4− or GTPγS (to mimic the active state). Bound proteins were separated by SDS–PAGE and detected by autoradiography. (b) GST pull-down experiment in cell lysates demonstrating that the interactions between overexpressed dysbindin, GASP-1 and HRS were independent of the GST–Gαs activation state. Lysates from HEK293 cells transiently transfected with HRS, Myc–dysbindin or Cherry–GASP-1 were incubated with GST alone, inactive GST–Gαs–GDP or active GST–Gαs–GDP/AlF4− or GTPγS (as described in a). Bound proteins were analysed by immunoblotting (IB) for HRS, Myc (dysbindin) and Cherry (GASP-1). The interaction with Gβ1 served as a control to confirm the activation state of GST–Gαs because Gβ1 only binds to inactive Gαs. This control was included in each experiment in a,b. (c) The downstream effectors of Gαs were not involved in CXCR4 downregulation. Control and Gαs-depleted HEK293 cells expressing HA–CXCR4 were treated for 8 h with vehicle (DMSO), 10 μM forskolin (an adenylyl cyclase activator) or 10 μM H89 (a PKA inhibitor). The steady-state levels of HA–CXCR4 were analysed by immunoblotting using the indicated antibody. (d) Quantification of the HA–CXCR4 levels in c. The data are presented as the percentage of HA–CXCR4 compared with control siRNA cells treated with DMSO. The data are presented as the mean±s.e.m. of ≥3 independent experiments and statistical analysis was performed using the Student’s t-test. **P≤0.01.
