title: 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 gamma-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 beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(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.
authors.affiliation: Cell Biology Research Group, Robarts Research Laboratory and Department of Physiology and Pharmacology, The University of Western Ontario, 100 Perth Drive, London, Ontario N6A 5K8, Canada.
authors.forename: Gurpreet K
authors.initials: GK
authors.lastname: Dhami
authors.forename: Andy V
authors.initials: AV
authors.lastname: Babwah
authors.forename: Rachel
authors.initials: R
authors.lastname: Sterne-Marr
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007295:Inositol Phosphates / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016318:Quisqualic Acid / Q000494:pharmacology; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D007295:Inositol Phosphates; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; C104077:metabotropic glutamate receptor type 1; D016318:Quisqualic Acid; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; C496569:Grk2 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D008239:Lysine; D000409:Alanine; D002118:Calcium