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 journalArticle

8 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Cell Physiology
Volume279
Issue number6 48-6
StatePublished - 2000

Fingerprint

Cholecystokinin Receptors
Phosphorylation
G-Protein-Coupled Receptors
Phosphoserine
Guanine Nucleotides
Endosomes
Recycling
Cricetulus
Occupations
GTP-Binding Proteins
Aspartic Acid
Serine
Ovary
Carrier Proteins
Cells
Ligands
Cell Line
Kinetics

Keywords

  • Endocytosis
  • G protein-coupled receptor
  • Receptor phosphorylation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking. / Rao, Rammohan V.; Holicky, Eileen L.; Kuntz, Susan M.; Miller, Laurence J.

In: American Journal of Physiology - Cell Physiology, Vol. 279, No. 6 48-6, 2000.

Research output: Contribution to journalArticle

@article{2edac75a161949f8b247f4c267ae97c1,
title = "CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking",
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.",
keywords = "Endocytosis, G protein-coupled receptor, Receptor phosphorylation",
author = "Rao, {Rammohan V.} and Holicky, {Eileen L.} and Kuntz, {Susan M.} and Miller, {Laurence J}",
year = "2000",
language = "English (US)",
volume = "279",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "6 48-6",

}

TY - JOUR

T1 - CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

AU - Rao, Rammohan V.

AU - Holicky, Eileen L.

AU - Kuntz, Susan M.

AU - Miller, Laurence J

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

KW - Endocytosis

KW - G protein-coupled receptor

KW - Receptor phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=0033637172&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033637172&partnerID=8YFLogxK

M3 - Article

VL - 279

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 6 48-6

ER -