Topology of the signal transduction of the G protein-coupled somatostatin receptor sst2 in human glioma cells

By a dual approach, using electron microscopy and biochemical techniques, we investigated the topology of the somatostatin receptor sat, with its inhibitory C protein Gi alpha after ligand-induced stimulation and internalization in human glioma cells. On intact cells, the sst(2) was labeled at 80 degrees by an antibody directed to its extracellular sequence followed by a 15-nm gold-labeled secondary antibody. In the presence of the ligand, internalization was induced by exposure to 37 degreesC for 5-10 min. Then, cells were either fixed for immunoelectron-microscopic analysis or homogenized for density gradient separation. After post-embedding staining of the sst(2)-labeled sections with anti-Gi alpha (1-3) or anti-caveolin, a co-localization of sst(2), Gi alpha and caveolin was detected in endosomal vesicles after 5 min of internalization, but not after 10 min. Furthermore, the gold-labeled organelles containing the internalised receptor were separated from the non-labeled ones on sucrose gradients (density shift separation) and analyzed by Western blotting. Also here, in fractions with higher densities, sst(2) could be co-stained with Gi alpha and caveolin after 5 min. From these congruent results from both methods, it can be concluded that, in human glioma cells, the receptor sst(2) (1) is internalised in caveolin-positive vesicles and (2) is neighboured to its Gi alpha proteins at the plasma membrane and early endosomes.