Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail

Citation
N. Hukovic et al., Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail, J BIOL CHEM, 274(35), 1999, pp. 24550-24558
Citations number
36
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
35
Year of publication
1999
Pages
24550 - 24558
Database
ISI
SICI code
0021-9258(19990827)274:35<24550:AUOHSR>2.0.ZU;2-J
Abstract
We have previously reported that the human somatostatin receptor type 1 (hS STR1) stably expressed in Chinese hamster ovary-Iii cells does not internal ize but instead up-regulates at the membrane during continued agonist treat ment (1 mu M somatostatin (SST)-14 x 22 h), Here we have investigated the m olecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in int act cells but not in membrane preparations. Although hSSTR1 was acutely des ensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continue d agonist exposure (22 h) restored functional effector coupling. Up-regulat ion was unaffected by cycloheximide but blocked by okadaic acid. Confocal f luorescence immunocytochemistry of intact and permeabilized cells showed pr ogressive, time-dependent increase in surface hSSTR1 labeling, associated w ith depletion of intracellular SSTR1 immunofluorescent vesicles. To investi gate the structural domains of hSSTR1 responsible for up-regulation, we con structed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 recepto rs, Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type h SSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ab ility to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 ind uced 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 s how that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dep endent active process of receptor recruitment from a pre-existing cytoplasm ic pool to the plasma membrane. It does not require new protein synthesis o r signal transduction, is sensitive to dephosphorylation events, and critic ally dependent on molecular signals in the receptor C-tail.