RT Journal Article
SR Electronic
T1 Agonist-stimulated phosphorylation of the carboxyl-terminal tail of the secretin receptor.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 818
OP 824
VO 48
IS 5
A1 F Ozcelebi
A1 M H Holtmann
A1 R U Rentsch
A1 R Rao
A1 L J Miller
YR 1995
UL http://molpharm.aspetjournals.org/content/48/5/818.abstract
AB The secretin receptor belongs to a recently recognized family of G protein-coupled receptors that lack the sequence motifs typical of the beta-adrenergic receptor family. Because our understanding of the regulatory mechanisms for these receptors is largely based on the latter group, we have begun to explore these mechanisms in the secretin receptor. In the present study, we focused on receptor phosphorylation, a key mechanism of receptor desensitization. Secretin receptor phosphorylation was demonstrated in intact transiently transfected COS cells and a stable receptor-bearing Chinese hamster ovary cell line in response to stimulation with native agonist. Secretin phosphoreceptor migrated on a sodium dodecyl sulfate-polyacrylamide gel at M(r) 57,000-62,000 in its native state and at M(r) 42,000 after deglycosylation, similar to the receptor that had been affinity-labeled with 125I-[Tyr10,p-NO2-Phe22]-secretin-27. Phosphorylation occurred rapidly in a secretagogue concentration-dependent manner, with 0.1 microM secretin eliciting a 7.2-fold increase in phosphorylation after 2 min. One-dimensional phosphopeptide mapping after cyanogen bromide cleavage revealed a single band of M(r) 9400, corresponding in size to the carboxyl-terminal tail domain. This identification was confirmed with a truncation mutant in which potential sites of phosphorylation in the tail were eliminated and no agonist-stimulated phosphorylation was observed. Phosphoamino acid analysis of the secretin phosphoreceptor demonstrated predominance of phosphothreonine over phosphoserine (3.2:1), with no phosphotyrosine observed. Three distinct carboxyl-terminal truncation mutants were constructed to each eliminate a subset of potential phosphorylation sites, and differential levels of phosphorylation were observed. Appropriate biosynthetic processing, expression on the cell surface, and signaling for each of these constructs were ensured by demonstration of ligand binding and cAMP responsiveness. Thus, receptors in the recently described secretin receptor family are phosphorylated in response to agonist stimulation in a manner analogous to the beta-adrenergic receptor, likely representing an important molecular mechanism for receptor desensitization.