CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

Rammohan V. Rao, Eileen L. Holicky, Susan M. Kuntz, Laurence J. Miller

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Original languageEnglish (US)
Pages (from-to)C1986-C1992
JournalAmerican Journal of Physiology - Cell Physiology
Volume279
Issue number6 48-6
DOIs
StatePublished - 2000

Keywords

  • Endocytosis
  • G protein-coupled receptor
  • Receptor phosphorylation

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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