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 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.
Kreienkamp
H J
HJ
Institut für Zellbiochemie und klinische Neurobiologie, Universität Hamburg, Germany.
Roth
A
A
Richter
D
D
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D015221:Potassium Channels / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D017481:Receptors, Somatostatin / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013004:Somatostatin / Q000494:pharmacology*; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism; D013912:Threonine; D014162:Transfection; D014982:Xenopus laevis
D018076:DNA, Complementary; D008565:Membrane Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; C103631:somatostatin receptor 3; C098681:somatostatin receptor subtype-4; D005576:Colforsin; D013912:Threonine; D013004:Somatostatin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases