Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Abstract

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and ␥-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation-and ␤-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and G␣ q/11 . G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys 691 or Lys 692 prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys 692 prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.Metabotropic glutamate receptors (mGluRs) 1 comprise a family of eight G protein-coupled receptors (GPCRs) that are activated by the excitatory amino acid glutamate and play an important role in regulating neuronal development and synaptic plasticity (1-3). Group I mGluRs (mGluR1 and mGluR5) are coupled via the heterotrimeric G protein G␣ q/11 to the activation of phospholipase C, resulting in the formation of both inositol 1,4,5-triphosphate and diacylglycerol, as well as increases in intracellular Ca 2ϩ concentrations. The alternative gene splicing of mGluR1 generates five carboxyl-terminal domain mGluR1 splice variants (1a, 1b, 1c, 1d, and 1e) (1-4). The G protein-coupling domain for these slice variants is conserved, and it is mediated by amino acid residues localized within the second and third intracellular loops and the membrane proximal region of the mGluR1 carboxyl-terminal tail (4).Similar to most GPCRs, mGluR activity is regulated by serine/threonine protein kinases (5-9). In general, the desensitization of GPCRs involves either phosphorylation by second messenger-dependent protein kinase or phosphorylation by G protein-coupled receptor kinases (GRKs) to promote the binding of arrestin proteins (10, 11). GRK2, GRK4, and GRK5 have each been demonstrated to contribute to the desensitization and internalization of mGluR1 in both heterologous cell cultures and Purkinje cells (5)(6)(7)(12)(13)(14)(15). Although GRK2 and GRK4 promote the ␤-arrestin-dependent internalization of mGlu...

M3 Muscarinic Acetylcholine Receptor-Mediated Signaling Is Regulated by Distinct Mechanisms

“…The crystal structure of GRK2 (Tesmer et al, 2005) suggests that it may simultaneously sequester both active G␣ q and free G␤␥, which, in addition to receptor phosphorylation, may increase the strength and effectiveness of GRK2-mediated receptor regulation. We and others have demonstrated that GRK2-regulated GPCRs, such as the H1 histamine (Iwata et al, 2005), M 1 mAChR (Willets et al, 2005), metabotropic glutamate (Dhami et al, 2005), and A, cells were transfected with SMARTpool siRNA and harvested 72 h later. Blots were incubated with a monoclonal antibody for arrestin-2 that cross-reacts with arrestin-3.…”

Somatic Mutations in GRM1 in Cancer Alter Metabotropic Glutamate Receptor 1 Intracellular Localization and Signaling

“…GRK2-mediated attenuation of mGluR1a signaling occurs as the consequence of the concomitant association of the kinase with the second intracellular loop domain of the receptor and Gaq (Dhami et al, 2004(Dhami et al, , 2005Dhami and Ferguson, 2006;Ferguson, 2007). We investigated the effect of three intracellular loop 2 SNVs on the association of GRK2 with mGluR1a.…”

“…Desensitization of mGluRs is complex as mGluR1a desensitization includes phosphorylationdependent and -independent mechanisms (Dhami et al, 2004(Dhami et al, , 2005Dhami and Ferguson, 2006;Ferguson, 2007;Ribeiro et al, 2009). G protein-coupled receptor kinase 2 (GRK2)-mediated mGluR1/5 desensitization is phosphorylation independent, whereas second-messenger-dependent kinases phosphorylate the second intracellular loop and carboxyl-terminal tail domains of mGluR1a to mediate phosphorylation-dependent mGluR desensitization (Francesconi and Duvoisin, 2000).…”

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

“…Binding of GRK2 to G␣q/11 effectively terminates transduction of the G␣ signal (3, 5-7). This phosphorylation-independent mechanism has now been implicated in the regulation of several Gq/11-coupled receptor signaling events (2,8,9).…”

“…Binding of GRK2 to G␣q/11 effectively terminates transduction of the G␣ signal (3, 5-7). This phosphorylation-independent mechanism has now been implicated in the regulation of several Gq/11-coupled receptor signaling events (2,8,9).Amiloride-sensitive epithelial Na ϩ channels (ENaC) play an important role in regulating Na ϩ and fluid homeostasis, maintaining blood pressure (10) and regulating volume of the alveolar fluid (11). ENaC is expressed in a variety of tissues including the renal collecting duct, the distal colon, the lungs and the ducts of exocrine glands.…”