Rat Somatostatin Receptor Subtype 4 Can Be Made Sensitive to Agonist-Induced Internalization by Mutation of a Single Threonine (Residue 331)

Abstract

A sequence motif of 20 amino acid residues within the C-terminal portion of the rat somatostatin receptor subtype 4 (SSTR4) has been shown to prevent rapid agonist-dependent receptor internalization in transfected human embryonic kidney (HEK) cells. Molecular dissection of this motif by biochemical ligand-binding assays revealed that the block was released by mutating a single residue (threonine 331) to an alanine. These data are in line with confocal microscopic analysis of cultured primary neurons microinjected with cDNA constructs encoding either SSTR4 or the mutant T331A. Immunocytochemical analysis showed that the mutant receptor, but not SSTR4, was internalized. However, internalized T331A was not recycled to the cell surface, suggesting that it lacks sequence elements that determine intracellular sorting after endocytosis. Neither wildtype SSTR nor the mutant T331A exhibited functional desensitization when assayed for their ability to inhibit adenylate cyclase. In agreement with this, the wt receptor and its mutant were not phosphorylated 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 decayed only slowly after washout of the agonist. This is in contrast to another somatostatin receptor subtype, SSTR3, which mediates rapidly desensitizing currents. Binding experiments on HEK cells transfected with either SSTR3 or 4 indicated that this difference is not attributable to slow dissociation of the agonist from the receptor, suggesting that SSTR4 mediates long-lasting signalling, a property which may be relevant for clinical therapy.

Differential β-Arrestin Trafficking and Endosomal Sorting of Somatostatin Receptor Subtypes

“…onine residues occupying four of five positions in its carboxylterminal tail (13). In addition, we demonstrate that the sst 4 receptor was resistant to agonist-induced phosphorylation and ␤-arrestin-dependent trafficking even in the presence of GRK2.…”

“…These results imply either peculiar mechanisms of receptor desensitization or resistance to desensitization. In fact, it has previously been shown that the rat sst 4 receptor fails to internalize after agonist activation (13). Consistent with the previous findings, we show that sst 4 did not undergo any detectable phosphorylation after agonist activation despite the presence of a number of potential phosphate acceptor sites including a cluster of thre- 3 was used to examine colocalization between a 30-min agonist pulse of antibody-labeled receptors (red) followed by a 20-min chase with transferrin (green) in the absence of SS-14 as described under "Experimental Procedures."…”

“…All polyclonal rabbit antisera were purified by affinity chromatography using the Sulfo-Link coupling gel coupled to the appropriate immunizing peptide according to the instructions of the manufacturer (Pierce). Plasmid constructs of the rat sst 1 , sst 2A , sst 3 , sst 4 , and sst 5 containing the amino-terminal T7 epitope tag sequence MASMTGGQQMG in pcDNA3 have been described previously (13,15,21). Truncated sst 2A mutants were created by PCR using primers that introduce stop codons at suitable positions (22).…”

“…So far only species-related differences in agonistmediated endocytosis of somatostatin receptors have been observed, e.g. the rat sst 4 receptor appears to be largely resistant to agonist-induced internalization and desensitization (13), whereas the human sst 4 receptor has been reported to undergo a very slow but clearly detectable endocytosis. Other studies have shown that the human sst 1 receptor but not the rat sst 1 receptor failed to internalize in an agonist-dependent manner (14,15).…”

Antidepressants Influence Somatostatin Levels and Receptor Pharmacology in Brain

“…In contrast, the changes in the CA1 region were due to the sst 1/4 receptors. The sst 1 and sst 4 subtypes are found in the hippocampus but unlike the sst 2 subtype were shown to have poor internalization yields (Nouel et al, 1997;Kreienkamp et al, 1998;Stroh et al, 2000). In a recent publication new evidence is provided showing that sst 1 mediates agonist-induced endocytosis, recycling and reendocytosis of its cognate ligand (Roosterman et al, 2007) 4 alone, something that we cannot examine due to lack of specific radioligands.…”