title: Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.
abstract: In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins.
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Mundell
authors.forename: Giordano
authors.initials: G
authors.lastname: Pula
authors.forename: Julia C A
authors.initials: JC
authors.lastname: More
authors.forename: David E
authors.initials: DE
authors.lastname: Jane
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roberts
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
mesh_terms: D019390:Arrestins / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D019390:Arrestins; D010750:Phosphoproteins; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases