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 M somatostatin (SST)-14 ؋ 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 (⌬) 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, ⌬ 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.

Agonist-induced Phosphorylation of Somatostatin Receptor Subtype 1 (Sst1)

“…However, the relationship between desensitization and receptor internalization was not examined in this cell line (35). In another study, the human sst1 receptor expressed in CHO-K1 cells was shown to desensitize after treatment with SRIF for 60 min when little receptor internalization had occurred (36,39). We show here that desensitization of the native rat sst1 receptor proceeds extremely rapidly in CHO-K1 cells, with a half-time less than 2 min.…”

“…Pretreatment of cells with SRIF leads to desensitization of both the rat and human sst1 receptor (35,39). In HEK cells expressing rat sst1-tag no effect was observed after a 10-min pretreatment with 1 M SRIF but significant desensitization occurred after preincubation for 30 and 120 min (35).…”

“…A previous report showing that a substantial portion of sst1 receptors are localized intracellularly in unstimulated CHO cells (39) suggested that the fraction of receptors resistant to phosphorylation may derive from this cytoplasmic pool. To determine whether the phosphorylated and phosphorylation resistant forms of sst1 were present in different intracellular compartments, we exploited the specificity of the carbohydrate selective endoglycosidase, Endo H. This enzyme can differentiate between immature receptors in the ER which contain high mannose carbohydrates and are sensitive to Endo H digestion and fully processed receptors containing complex N-linked oligosaccharides which are Endo H resistant (42).…”

Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells

“…Exposure of agonists to G protein-coupled receptors frequently results in downregulation of the receptor transcript levels, but receptor upregulation has also been demonstrated (Siegrist et al 1994, Schanstra et al 1998, Froidevaux & Eberle 2002, Ankö & Panula 2006. Inoue et al (1999) found that calcitonin downregulates calcitonin receptor mRNA in mouse bone marrow cells and Dupre et al (2003) report the ligand-induced downregulation of the PAF-R. On the other hand, gene expression of the human somatostatin receptor type I is actively upregulated by somatostatin (Hukovic et al 1999). The regulation of CL/RAMP2 mRNA levels by AM in HMECs might reflect a physiological adaptation, which enables the cells to adjust the sensitivity of receptor-mediated processes by changes in receptor number, and, therefore, according to the level of receptor activation.…”

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

“…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).…”