Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.

Endocytosis of somatostatin receptors could regulate cellular responses to the two natural peptides, somatostatin-14 and somatostatin-28, and to synthetic ligands used in the clinical diagnosis and symptomatic therapy of neuroendocrine tumours. The five cloned SSTRs with or without epitope tags at their carboxyl-termini were expressed in rat insulinoma 1046-38 cells. Application of the two natural peptides or octreotide, 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 decrease of cell surface binding-sites for 125I-Tyr11-somatostatin-14. In contrast, cells transfected with subtype 5 cDNA only responded to stimulation with octreotide or somatostatin-28. Cells transfected with subtype 1 cDNA responded to somatostatin-14 and 28, while cells expressing subtype 4 cDNA showed no response. Confocal microscopy revealed that 6 min after stimulation with somatostatin-14 at 37 degrees C, tagged somatostatin receptor subtypes 1, 2 and 3 were internalized into vesicles. Internalization was not observed at 4 degrees C in the presence of 0.4 M sucrose and 80 microM phenylarsine oxide and hence proceeded via endocytosis through clathrin-coated pits and vesicles. After 20 min the internalized receptors appeared in perinuclear vesicles and after 120 min they reappeared at the plasma membrane. This recycling was not sensitive to cycloheximide and, hence, not dependent on de novo protein synthesis. Recovery of cell surface receptors was, however, inhibited by brefeldin A, monensin and bafilomycin A1, indicating that receptor recycling proceeded through vesicular traffic of acidified compartments. The data are consistent with the assumption that the observed agonist and subtype specific internalization of somatostatin receptors in a neuroendocrine cell line may be important for tumour diagnosis and therapy and, thus, suggest a manifold control in cellular signalling.

Here we report on the molecular mechanism responsible for the endocytotic process by analyzing the agonist-dependent phosphorylation of wild-type and mutant receptors expressed in human embryonic kidney cells. Wild-type SSTR3 is phosphorylated in response to agonist treatment. Phosphorylation is markedly reduced in a S341A/ S346A/S351A triple mutant and is also reduced, but to a lesser extent, in the T357A point mutant. Internalization of the wild-type receptor is preceded by a functional desensitization of the receptor; in contrast, the triple serine mutant does not desensitize after treatment with agonists as assayed by its ability to inhibit forskolinstimulated adenylate cyclase activity. After internalization via a clathrin-coated vesicle mediated endocytotic pathway, SSTR3 efficiently recycles to the cell surface, suggesting that agonist mediated endocytosis is necessary for the functional resensitization of a phosphorylated and desensitized receptor.Prolonged exposure of G protein-coupled receptors (GPCRs) 1 to their agonists frequently causes a diminution of the initial response. This phenomenon has been termed desensitization, and it is often associated with phosphorylation of the receptor protein followed by binding of the regulatory protein ␤-arrestin (1, 2). The receptor is then internalized into an intracellular compartment where it is inaccessible to hydrophilic ligands.␤-Arrestin has been proposed to function as an adaptor between phosphorylated receptors and clathrin cages, thus directing receptors to the endocytotic pathway (3, 4). In the acidified environment of endosomes, the ligand is removed and the receptor is dephosphorylated and recycled to the cell surface (5, 6).In the case of receptors for the neuropeptide somatostatin (SST), the processes of desensitization and internalization are of considerable clinical importance as stable peptide analogs of somatostatin are used for the long term treatment of hypersecretory neuroendocrine tumors of the pituitary and pancreas (7). These analogs not only inhibit hormone secretion, but also have an antiproliferative effect on tumor cells (8). They are also used for the preoperative localization of tumors by SSTR scintigraphy (9), a process which presumably relies on the internalization of the radioactive ligand by its target cells.The fact that somatostatin receptors and their ligands are internalized was originally disputed (10), but recently it was shown that several SSTR-expressing tumor cells do internalize radioactive SST analogs (11). We have recently shown that in human embryonic kidney cells expressing individual rat SSTR subtypes (rSSTR1-5), SSTR1-3 are rapidly internalized in the presence of either of the two naturally occurring agonists, SST14 and SST28. SSTR5 is internalized only in the presence of SST28, whereas SSTR4 is not internalized by either agonist (12, 13). Studies on the internalization of human and mouse SSTRs confirmed that internalization is subtype-selective, although some species-specific differences were reported (14, 15). ...

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.

Aldehyde hydrates are important but highly unstable, transient intermediates in biological and synthetic oxidations to carboxylic acids. We here report N-oxides as the first class of chemical reagents capable of stabilizing such water adducts. This stabilizing effect (studied in solution and in the solid state) seems to be based on the formation of hydrogen bonds.