Phosphorylation Uncouples the Gastrin-releasing Peptide Receptor from Gq

Abstract

Previous work on the desensitization of G proteincoupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G q -coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of ϳ1 mol PO 4 /mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V max ) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the ␣ q or ␤␥ subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.Bombesin-like peptides elicit a variety of effects including mitogenesis, hormone secretion, and modulation of neuron firing rate (1). These effects are transduced through a family of G protein-coupled receptors (GPCRs) 1 including the gastrin-releasing peptide receptor (GRP-R) (2, 3). An agonist-activated GPCR catalyzes the exchange of guanine nucleotide on the ␣ subunit of a heterotrimeric G protein leading to the formation of G␣-GTP. The subsequent dissociation of the heterotrimeric G protein leads to stimulation of signal transduction cascades mediated by the free G␣-GTP and G␤␥ subunits (4). In the bombesin receptor family, the activated GRP-R catalyzes guanine nucleotide exchange on G␣ q (5) to activate effectors including phospholipase C-␤ (3).Mechanisms to attenuate receptor signaling have evolved that limit the amplitude and/or duration of the signal transduction cascade(s) and are collectively referred to as "desensitization." At the molecular level, desensitization may result from the degradation of ligand, from changes in receptor availability or activity, or from changes in the availability or activity of downstream effector molecules (6). Rhodopsin, which signals through transducin (G t ), and the ␤ 2 -adrenergic receptor (␤ 2 AR), which signals through G s , have been the most extensively studied GPCRs. In both cases, rapid agonist-induced receptor phosphorylation along with subsequent binding of an arrestin to the receptor play critical roles in receptor deactivation (7,8). Many other GPCRs are also known to be rapidly phosphorylated after addition of agonist (9, 10), including the GRP-R (11). Two classes of protein kinase have been implicated in agonist-induced GPCR phosphorylation: 1) the second messenger-dependent protein kinases A and C and 2) the second messenger independent kinases, called G protein-coupled receptor kinases (GRKs), including rhodopsin kinase (RK or GRK1) an...

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

“…GRPr ligand binding sites in membrane preparations were quantitated by analysis of the displacement of the specific GRPr antagonist 125 I-ME by ME or displacement of the GRPr agonist 125 I-697 by D-Phe 6 -697 as de- scribed previously for 125 I-ME (Kroog et al, 1999). Results were similar with both radioligands (data not shown).…”

“…Intact Cell Phosphorylation. GRPr phosphorylation assays in whole cells were performed using published procedures (Kroog et al, 1999). Briefly, cells were incubated for 3 h at 37°C in phosphate-free media with 5% dialyzed FBS and 50 Ci of 32 PO 4 per milliliter (PF media) and then treated as described in the figure legends.…”

“…In vitro, GRPr is a substrate for phosphorylation by GRK2. Both in vitro GRK2-phosphorylated GRPr and agonist-phosphorylated GRPr prepared from intact cells show approximately 75 to 80% reduction in the rate of catalysis of guanine nucleotide exchange on G q in an in vitro reconstitution assay in the absence of arrestin (Kroog et al, 1999). However, the importance of these findings for GRPr desensitization in cells is unknown.…”

“…Published methods (Hellmich et al, 1997) were used to prepare GRPr containing postnuclear (P2) membranes from cells expressing high levels of GRPr. 7M urea-extracted membranes were prepared as described previously (Kroog et al, 1999).…”

“…Several classes of protein kinase have been implicated in GPCR phosphorylation: 1) second messenger-dependent protein kinases A (PKA) and C; 2) second messenger-independent kinases, called G protein-coupled receptor kinases (GRKs), which include rhodopsin kinase (GRK1) and ␤-adrenergic receptor kinase 1 (GRK2); and 3) casein kinase 1␣ (Premont et al, 1995;Tobin et al, 1997;Krupnick and Benovic, 1998). Many GPCRs, including GRPr, are known to be substrates for phosphorylation by GRKs in vitro (Krupnick and Benovic, 1998;Kroog et al, 1999). Several are also proven substrates for phosphorylation by second messenger-dependent kinases (Kelleher and Johnson, 1986;Richardson et al, 1992).…”