Metabotropic Glutamate Receptor 1 Internalization Induced by Muscarinic Acetylcholine Receptor Activation: Differential Dependency of Internalization of Splice Variants on Nonvisual Arrestins

• Published in: Molecular pharmacology Vol. 61; no. 5; pp. 1114 - 1123
• Main Authors: Mundell, Stuart J., Matharu, Anne-Lise, Pula, Giordano, Holman, David, Roberts, Peter J., Kelly, Eamonn
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2002
• Subjects: 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide; 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester; acetylcholine receptor; AChR; Alternative Splicing - drug effects; Amino Acid Sequence; analysis of variance; Animals; ANOVA; arr-DNM; arrestin-2 dominant-negative mutant; Arrestins - metabolism; bovine serum albumin; BSA; Ca2+-calmodulin-dependent kinase II; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; CaMKII; Cells, Cultured; Cercopithecus aethiops; COS Cells; CPCCOEt; DMEM; Dulbecco’s modified Eagle’s medium; dyn-DNM; dynamin dominant-negative mutant; Dynamins; ELISA; Endocytosis - drug effects; Endocytosis - physiology; enzyme-linked immunosorbent assay; G-protein-coupled receptor; G-protein-coupled receptor kinase; GF 109203X; GFP; Glutamic Acid - pharmacology; GPCR; green fluorescent protein; GRK; GTP Phosphohydrolases - metabolism; HEK; hemagglutinin; human embryonic kidney; Humans; inositol phosphate; kilobase(s); KN-93; metabotropic glutamate receptor; mGluR; Molecular Sequence Data; N-(2-[N-[4-chlorocinnamyl]-N-methylaminomethyl]phenyl)-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide; PCR; phorbol 12-myristate 13-acetate; PKA; PKC; PMA; polymerase chain reaction; protein kinase A; protein kinase C; Protein Kinase C - metabolism; Receptors, Metabotropic Glutamate - genetics; Receptors, Metabotropic Glutamate - metabolism; Receptors, Muscarinic - metabolism; Second Messenger Systems; Signal Transduction - physiology; TBS; Transfection; Tris-buffered saline; GRK; KN-93; dyn-DNM; TBS; CPCCOEt; PMA; IP; PKA; PKC; mGluR; HEK; GFP; DMEM; BSA; GF 109203X; AChR; CaMKII; ANOVA; kb; arr-DNM; GPCR; HA; PCR; ELISA
• ISSN: 0026-895X
• DOI: 10.1016/S0026-895X(24)12205-5

In this study, we characterized the glutamate- or second-messenger kinase-dependent internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M1 muscarinic receptors in human embryonic kidney 293 cells, induced the internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca2+ calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.