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 sst2 with its inhibitory G protein Gialpha after ligand-induced stimulation and internalization in human glioma cells. On intact cells, the sst2 was labeled at 8 degrees C 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 degrees C 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 sst2-labeled sections with anti-Gialpha1- 3 or anti-caveolin, a co-localization of sst2, Gialpha 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, sst2 could be costained with Gialpha and caveolin after 5 min. From these congruent results from both methods, it can be concluded that, in human glioma cells, the receptor sst2 (1) is internalised in caveolin-positive vesicles and (2) is neighboured to its Gialpha proteins at the plasma membrane and early endosomes.
Anatomisches Institut der Universität Kiel, Germany. rment@anat.uni-kiel.de
R
R
Mentlein
J
J
Held-Feindt
B
B
Krisch
D000818:Animals; D000906:Antibodies; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis*; D004705:Endocytosis / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis / Q000378:metabolism*; D005909:Glioblastoma; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D016253:Microscopy, Immunoelectron; D015282:Octreotide / Q000494:pharmacology; D015388:Organelles / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D017481:Receptors, Somatostatin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013004:Somatostatin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D000906:Antibodies; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D006728:Hormones; D017481:Receptors, Somatostatin; D013004:Somatostatin; C093733:somatostatin receptor 2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D015282:Octreotide