Immunoelectronmicroscopic Analysis of the Ligand-induced Internalization of the Somatostatin Receptor Subtype 2 in Cultured Human Glioma Cells

Abstract

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.

Receptor-mediated internalization of somatostatin in rat cortical and hippocampal neurons

“…These findings are consistent with earlier studies demonstrating the internalization of gold-conjugated SRIF-14 in human astrocytoma cells in culture via the sst 2A receptor (Feindt et al, 1995;Krisch et al, 1998). As in neurons, astroglial internalization was associated with the appearance of intracytoplasmic fluorescent clusters, although the latter were larger than those seen after internalization in nerve cells.…”

“…However, the sst 2A receptor subtype has been proposed to internalize in rat brain in response to endogenous ligand release, based on correlative immunocytochemical data between the distribution of SRIF axon terminals and sst 2A receptors (Dournaud et al, 1998). There is also electron microscopic evidence for ligand-induced internalization of the sst 2A receptor in human glioma cells (Krisch et al, 1998), but again nothing is known of the internalization of this or other sst receptor subtypes in nonmalignant astrocytes. The aim of the present study was therefore to determine whether SRIF agonists are indeed internalized in neurons and astrocytes of central origin and to investigate whether this internalization is mediated by sst 2A and/or other sst receptor subtypes.…”

Somatostatin and Somatostatin Receptors

“…When transfected in either CHO-K1, HEK-293, or even pancreatic b-cells, human and rat SSTR2 internalize in response to SST stimulation (Hukovic et al 1996;Roosterman et al 1997;Roth et al 1997b;Cescato et al 2006), via a clathrin-dependent pathway. Furthermore, endocytosis of SSTR2 was also demonstrated in glioma and neuroblastoma cells that endogenously express the receptor (Koenig et al 1997;Krisch et al 1998). In other cell types, the densitization, internalization, and phosphorylation of rat SSTR2 was observed (Hipkin et al 1997(Hipkin et al , 2000Roosterman et al 1997).…”

The Long and the Short Cycle

“…Vasopressin (1) is a small peptide hormone recognized by three different G protein-coupled receptors (GPCRs) 1 of which the V1a and V1b subtypes are coupled by G q/11 and the V2 is coupled to G s . Interaction with the agonist instantly leads to GPCR activation, phosphorylation, desensitization, and sequestration, followed by the return of the dephosphorylated receptors to the cell surface.…”

“…The pathway by which GPCRs are recycled is under intense study, and an increasing number of elements specific for the receptor or the cell type have been identified, although little is known about how the different pathways and organelles are involved. The majority of GPCRs internalize via clathrin-coated pits, although some, such as the somatostatin 2 receptor, have been found mostly in uncoated vesicles (1). Furthermore, in the case of the ␤ 2 adrenergic receptor (␤ 2 -AR), the endothelin receptor, and the cholecystokinin receptor, an additional internalization mechanism has been described involving caveolin (2-4).…”