Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent.
At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.
Mundell
S J
SJ
Department of Pharmacology, School of Medical Sciences, University of Bristol, Bristol, UK.
Matharu
A L
AL
Pula
G
G
Roberts
P J
PJ
Kelly
E
E
D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D019393:Arrestin; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; C104077:metabotropic glutamate receptor type 1; D018698:Glutamic Acid; D020558:GTP Phosphohydrolases; D034281:Dynamins