Agonist- and Protein Kinase C-Induced Phosphorylation Have Similar Functional Consequences for Gastrin-Releasing Peptide Receptor Signaling via Gq

• Published in: Molecular pharmacology 2003-10, Vol.64 (4), p.890-904
• Main Authors: Ally, Roxanne A., Ives, Kirk L., Traube, Elie, Eltounsi, Iman, Chen, Pei-Wen, Cahill, Patrick J., Battey, James F., Hellmich, Mark R., Kroog, Glenn S.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Arrestin - metabolism; Biological Transport; Bombesin - metabolism; Calcium - metabolism; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Phosphorylation; Protein Kinase C - metabolism; Protein Structure, Tertiary; Receptors, Bombesin - metabolism; Signal Transduction
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.64.4.890

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 Gq. 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-Gq 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 Gq signaling.