Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs)

Citation
Pj. Flannery et Rf. Spurney, Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), BIOCH PHARM, 62(8), 2001, pp. 1047-1058
Citations number
43
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
8
Year of publication
2001
Pages
1047 - 1058
Database
ISI
SICI code
0006-2952(20011015)62:8<1047:DOTPH(>2.0.ZU;2-Y
Abstract
To investigate the domains of the parathyroid hormone (PTH) receptor requir ed for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group -containing amino acids at the C-terminus. In mutant #2, we replaced threon ines 392 and 399 in the third intracellular loop with glycines. Co-transfec tion of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK 4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhib ition of PTH receptor signaling was associated with enhanced phosphorylatio n receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cA MP generation by the wild-type receptor and mutant #1, and caused phosphory lation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agon ist-induced phosphorylation of mutant #2 compared with that of either the w ild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was a ssociated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 fa iled to cause receptor desensitization and inhibited receptor internalizati on. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intrac ellular loop are not required for agonist-induced phosphorylation of PTH re ceptors, but are critical for both agonist-induced internalization of PTH r eceptors and GRK2-mediated regulation of PTH receptor signaling. (C) 2001 E lsevier Science Inc. All rights reserved.