title: Metabotropic glutamate receptor 1 internalization induced by muscarinic acetylcholine receptor activation: differential dependency of internalization of splice variants on nonvisual arrestins.
abstract: 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 M(1) 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 Ca(2+) 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.
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: Anne-Lise
authors.initials: AL
authors.lastname: Matharu
authors.forename: Giordano
authors.initials: G
authors.lastname: Pula
authors.forename: David
authors.initials: D
authors.lastname: Holman
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roberts
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019556:COS Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D034281:Dynamins; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D019390:Arrestins; D018094:Receptors, Metabotropic Glutamate; D011976:Receptors, Muscarinic; C104077:metabotropic glutamate receptor type 1; D018698:Glutamic Acid; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D034281:Dynamins