Agonist- and protein kinase C-induced phosphorylation have similar functional consequences for gastrin-releasing peptide receptor signaling via Gq

Roxanne A. Ally, Kirk L. Ives, Elie Traube, Iman Eltounsi, Pei Wen Chen, Patrick J. Cahill, James F. Battey, Mark Hellmich, Glenn S. Kroog

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Acute desensitization of many guanine nucleotide-binding protein-coupled receptors (GPCRs) requires receptor phosphorylation and subsequent binding of an arrestin. GPCRs are substrates for phosphorylation by several classes of kinases. Gastrin-releasing peptide receptor (GRPr) is phosphorylated by a kinase other than protein kinase C (PKC) after exposure to agonist and is also a substrate for PKC-dependent phosphorylation after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using GRPr mutants, we examined receptor domains required for agonist- and TPA-induced phosphorylation of GRPr and consequences of these phosphorylation events on GRPr signaling via G q. Agonist- and TPA-stimulated GRPr phosphorylation in cells require an intact carboxyl terminal domain (CTD). GRPr is phosphorylated in vitro by GPCR kinase 2 (GRK2) and multiple PKC isoforms. An intact DRY motif is required for agonist-stimulated phosphorylation in cells, and agonist-dependent GRK2 phosphorylation in vitro. Although GRPr CTD mutants do not show enhanced in vitro coupling to Gq relative to intact GRPr, CTD mutants have more potent Gq-dependent signaling in cells. Acute desensitization involves CTD-independent processes because desensitization can precede ligand binding in intact GRPr and CTD mutants. TPA-mediated impairment of GRPr-G q signaling in cells also requires an intact CTD. Similar to GRK2 phosphorylation, PKC phosphorylation reduces GRPr-Gq coupling by approximately 80% in vitro. Arrestin translocation to plasma membrane requires agonist, an intact DRY motif, and GRPr phosphorylation. Therefore, agonist- and PKC-induced GRPr phosphorylation sites are in nearby regions of the receptor, and phosphorylation at both sites has similar functional consequences for G q signaling.

Original languageEnglish (US)
Pages (from-to)890-904
Number of pages15
JournalMolecular Pharmacology
Volume64
Issue number4
DOIs
StatePublished - Oct 1 2003

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Bombesin Receptors
Protein Kinase C
Phosphorylation
Tetradecanoylphorbol Acetate
Guanine Nucleotides
Phosphotransferases
Arrestin
Carrier Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

Agonist- and protein kinase C-induced phosphorylation have similar functional consequences for gastrin-releasing peptide receptor signaling via Gq . / Ally, Roxanne A.; Ives, Kirk L.; Traube, Elie; Eltounsi, Iman; Chen, Pei Wen; Cahill, Patrick J.; Battey, James F.; Hellmich, Mark; Kroog, Glenn S.

In: Molecular Pharmacology, Vol. 64, No. 4, 01.10.2003, p. 890-904.

Research output: Contribution to journalArticle

Ally, Roxanne A. ; Ives, Kirk L. ; Traube, Elie ; Eltounsi, Iman ; Chen, Pei Wen ; Cahill, Patrick J. ; Battey, James F. ; Hellmich, Mark ; Kroog, Glenn S. / Agonist- and protein kinase C-induced phosphorylation have similar functional consequences for gastrin-releasing peptide receptor signaling via Gq In: Molecular Pharmacology. 2003 ; Vol. 64, No. 4. pp. 890-904.
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abstract = "Acute desensitization of many guanine nucleotide-binding protein-coupled receptors (GPCRs) requires receptor phosphorylation and subsequent binding of an arrestin. GPCRs are substrates for phosphorylation by several classes of kinases. Gastrin-releasing peptide receptor (GRPr) is phosphorylated by a kinase other than protein kinase C (PKC) after exposure to agonist and is also a substrate for PKC-dependent phosphorylation after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using GRPr mutants, we examined receptor domains required for agonist- and TPA-induced phosphorylation of GRPr and consequences of these phosphorylation events on GRPr signaling via G q. Agonist- and TPA-stimulated GRPr phosphorylation in cells require an intact carboxyl terminal domain (CTD). GRPr is phosphorylated in vitro by GPCR kinase 2 (GRK2) and multiple PKC isoforms. An intact DRY motif is required for agonist-stimulated phosphorylation in cells, and agonist-dependent GRK2 phosphorylation in vitro. Although GRPr CTD mutants do not show enhanced in vitro coupling to Gq relative to intact GRPr, CTD mutants have more potent Gq-dependent signaling in cells. Acute desensitization involves CTD-independent processes because desensitization can precede ligand binding in intact GRPr and CTD mutants. TPA-mediated impairment of GRPr-G q signaling in cells also requires an intact CTD. Similar to GRK2 phosphorylation, PKC phosphorylation reduces GRPr-Gq coupling by approximately 80{\%} in vitro. Arrestin translocation to plasma membrane requires agonist, an intact DRY motif, and GRPr phosphorylation. Therefore, agonist- and PKC-induced GRPr phosphorylation sites are in nearby regions of the receptor, and phosphorylation at both sites has similar functional consequences for G q signaling.",
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AU - Ives, Kirk L.

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AU - Eltounsi, Iman

AU - Chen, Pei Wen

AU - Cahill, Patrick J.

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AB - Acute desensitization of many guanine nucleotide-binding protein-coupled receptors (GPCRs) requires receptor phosphorylation and subsequent binding of an arrestin. GPCRs are substrates for phosphorylation by several classes of kinases. Gastrin-releasing peptide receptor (GRPr) is phosphorylated by a kinase other than protein kinase C (PKC) after exposure to agonist and is also a substrate for PKC-dependent phosphorylation after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using GRPr mutants, we examined receptor domains required for agonist- and TPA-induced phosphorylation of GRPr and consequences of these phosphorylation events on GRPr signaling via G q. Agonist- and TPA-stimulated GRPr phosphorylation in cells require an intact carboxyl terminal domain (CTD). GRPr is phosphorylated in vitro by GPCR kinase 2 (GRK2) and multiple PKC isoforms. An intact DRY motif is required for agonist-stimulated phosphorylation in cells, and agonist-dependent GRK2 phosphorylation in vitro. Although GRPr CTD mutants do not show enhanced in vitro coupling to Gq relative to intact GRPr, CTD mutants have more potent Gq-dependent signaling in cells. Acute desensitization involves CTD-independent processes because desensitization can precede ligand binding in intact GRPr and CTD mutants. TPA-mediated impairment of GRPr-G q signaling in cells also requires an intact CTD. Similar to GRK2 phosphorylation, PKC phosphorylation reduces GRPr-Gq coupling by approximately 80% in vitro. Arrestin translocation to plasma membrane requires agonist, an intact DRY motif, and GRPr phosphorylation. Therefore, agonist- and PKC-induced GRPr phosphorylation sites are in nearby regions of the receptor, and phosphorylation at both sites has similar functional consequences for G q signaling.

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