RAT SOMATOSTATIN RECEPTOR SUBTYPE-4 CAN BE MADE SENSITIVE TO AGONIST-INDUCED INTERNALIZATION BY MUTATION OF A SINGLE THREONINE (RESIDUE-331)

A sequence motif of 20 amino acid residues within the C-terminal porti on of the rat somatostatin receptor subtype 4 (SSTR4) has been shown t o prevent rapid agonist-dependent receptor internalization in transfec ted human embryonic kidney (HEK) cells. Molecular dissection of this m otif by biochemical ligand-binding assays revealed that the block was released by mutating a single residue (threonine 331) to an alanine, T hese data are in line with confocal microscopic analysis of cultured p rimary neurons microinjected with cDNA constructs encoding either SSTR 4 or the mutant T331A, Immunocytochemical analysis showed that the mut ant receptor, but not SSTR4, was internalized. However, internalized T 331A was not recycled to the cell surface, suggesting that it lacks se quence elements that determine intracellular sorting after endocytosis , Neither wildtype SSTR nor the mutant T331A exhibited functional dese nsitization when assayed for their ability to inhibit adenylate cyclas e, In agreement with this, the wt receptor and its mutant were not pho sphorylated in response to agonist treatment. Lack of desensitization of SSTR4 has been electrophysiologically verified by coexpressing the receptor with a G-protein-gated, inwardly rectifying potassium channel in Xenopus oocytes, A strong somatostatin 14 (SST14)-activated inward potassium current was observed that was long-lasting and which decaye d only slowly after washout of the agonist, This is in contrast to ano ther somatostatin receptor subtype, SSTR3, which mediates rapidly dese nsitizing currents. Binding experiments on HEK cells transfected with either SSTR3 or 4 indicated that this difference is not attributable t o slow dissociation of the agonist from the receptor, suggesting that SSTR4 mediates long-lasting signalling, a property which may be releva nt for clinical therapy.