Somatostatin receptors in Neuro2A neuroblastoma cells: Ligand internalization

1 Receptor-dependent internalization of somatostatin (SRIF) agonists has been a matter of controversy probably because [I-125]-Tyr(11)-SRIF-14 is rapidly degraded. We have studied the internalization of a stable somatostatin analogue, [I-125]-BIM-23027, in a neuronal cell line, Neuro2A, which natively expresses somatostatin sst(2) receptors. 2 Incubation of Neuro2A cells with [I-125]-BIM-23027 at 37 degrees C resulted in a time-dependent internalization of the ligand, which reached a maximum at 30 min. Acid-washing showed that cell-surface binding of the ligand accounted for only 34% of total binding at this time, internalization was dramatically reduced at 15 degrees C. 3 Internalization of [I-125]-BIM-23027 was prevented by inclusion of unlabelled somatostatin receptor agonists in a concentration-dependent manner. The IC50 values for inhibition of [I-125]-BIM-23027 internalization were approximately 100 fold lower than for inhibition of [I-125]-BIM-23027 binding to membrane homogenates but followed the same rank order of potencies. 4 Disruption of G-protein coupling by treatment with pertussis toxin caused a 60% reduction in internalization of ligand. A combination of antimycin (50 nM) and deoxyglucose (50 mM) pretreatment, which leads to a depletion of cellular ATP, decreased internalization of [I-125]-BIM-23027 by 66% of control and increased the proportion of surface-bound ligand. Hypertonic sucrose, which prevents clathrin-mediated endocytosis, reversibly abolished the internalization of ligand without increasing the proportion bound at the cell surface. 5 After internalization of [I-125]-BIM-23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37 degrees C. This finding suggests that the intracellular content of [I-125]-BIM-23027 reaches a steady state which is determined by the rates of both internalization and recycling of the ligand. In contrast to studies in which the internalization of [I-125]-Tyr(11)-SRIF-14 was examined, neither internalized nor recycled [I-125]-BIM-23027 was degraded to its component amino acids. 6 These findings indicate that the somatostatin agonist, [I-125]-BIM-23027, is internalized in a receptor-dependent manner which involves clathrin-coated pits in Neuro2A cells. Furthermore, much of the internalized ligand is rapidly recycled back to the extracellular medium without undergoing significant degradation.