Immunoelectronmicroscopic analysis of the ligand-induced internalization of the somatostatin receptor subtype 2 in cultured human glioma cells.
We analyzed the internalization of the receptor subtype 2 (sst2) for the neuropeptide somatostatin in glioma cells at the ultrastructural level using an antibody against an extracellular amino acid sequence. Intact cells derived from solid human gliomas or those of the human glioma cell line U343 were receptor-labeled (a) by classical gold immunocytochemistry using a 15-nm gold-labeled second antibody, (b) directly with the sst2 antibody adsorbed to 5-nm colloidal gold, and (c) with the physiological ligand somatostatin conjugated to 5-nm colloidal gold. The receptor was predominantly internalized via uncoated vesicles budding from the cell membrane but only rarely via coated pits, which has been mostly reported for G-protein-coupled, seven transmembrane-domain receptors. In the presence of ligand and sst2 antibody vesicles, tubule-like structures, and multivesicular bodies were labeled in superficial and in perinuclear portions of the cells within the first 30 min. Lysosomal labeling was observed after 30 min and especially after an hour of internalization time. This internalization route is also used to study the directly labeled sst2 antibody or the labeled ligand. However, the late endosomal compartment appears to be reached more rapidly in these latter experiments.
Department of Anatomy, University of Kiel, Kiel, Germany.
B
B
Krisch
J
J
Feindt
R
R
Mentlein
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism; D015153:Blotting, Western; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D016253:Microscopy, Immunoelectron; D051381:Rats; D011916:Rats, Inbred F344; D011956:Receptors, Cell Surface / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000378:metabolism*; D013004:Somatostatin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011956:Receptors, Cell Surface; D017481:Receptors, Somatostatin; D013004:Somatostatin; C093733:somatostatin receptor 2