title: Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail.
abstract: We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.
authors.affiliation: Fraser Laboratories, Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Hukovic
authors.forename: M
authors.initials: M
authors.lastname: Rocheville
authors.forename: U
authors.initials: U
authors.lastname: Kumar
authors.forename: R
authors.initials: R
authors.lastname: Sasi
authors.forename: S
authors.initials: S
authors.lastname: Khare
authors.forename: Y C
authors.initials: YC
authors.lastname: Patel
mesh_terms: D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004705:Endocytosis; D015200:Fluoroimmunoassay; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D017481:Receptors, Somatostatin / Q000819:agonists* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C101130:somatostatin receptor type 1; D019319:Okadaic Acid; C094573:somatostatin receptor 5; D003513:Cycloheximide; D000262:Adenylyl Cyclases