DISTINCT AGONIST-MEDIATED ENDOCYTOSIS OF CLONED RAT SOMATOSTATIN RECEPTOR SUBTYPES EXPRESSED IN INSULINOMA CELLS

Endocytosis of somatostatin receptors could regulate cellular response s to the two natural peptides, somatostatin-14 and somatostatin-28, an d to synthetic ligands used in the clinical diagnosis and symptomatic therapy of neuroendocrine tumours, The five cloned SSTRs with or witho ut epitope tags at their carboxyl-termini were expressed in rat insuli noma 1046-38 cells, Application of the two natural peptides or octreot ide, at 37 degrees C but not at 4 degrees C, to cells transfected with somatostatin receptor subtype 2 or 3 cDNA resulted in a significant d ecrease of cell surface binding-sites for I-125-Tyr(11)-somatostatin-1 4. In contrast, cells transfected with subtype 5 cDNA only responded t o stimulation with octreotide or somatostatin-28, Cells transfected wi th subtype 1 cDNA responded to somatostatin-14 and 28, while cells exp ressing subtype 4 cDNA showed no response. Confocal microscopy reveale d that 6 min after stimulation with somatostatin-14 at 37 degrees C, t agged somatostatin receptor subtypes 1, 2 and 3 were internalized into vesicles. Internalization was not observed at 4 degrees C in the pres ence of 0.4 M sucrose and 80 mu M phenylarsine oxide and hence proceed ed via endocytosis through clathrin-coated pits and vesicles, After 20 min the internalized receptors appeared in perinuclear vesicles and a fter 120 min they reappeared at the plasma membrane, This recycling wa s not sensitive to cycloheximide and, hence, not dependent on de nova protein synthesis, Recovery of cell surface receptors was, however, in hibited by brefeldin A, monensin and bafilomycin Al, indicating that r eceptor recycling proceeded through vesicular traffic of acidified com partments. The data are consistent with the assumption that the observ ed agonist and subtype specific internalization of somatostatin recept ors in a neuroendocrine cell line may be important for tumour diagnosi s and therapy and, thus, suggest a manifold control in cellular signal ling.