title: Agonist-induced desensitization and endocytosis of heterodimeric GABAB receptors in CHO-K1 cells.
abstract: gamma-Aminobutyric acid B (GABA(B)) receptor is the first discovered G protein-coupled receptor that requires two subunits, GB1 and GB2, to form a functional receptor. Whereas the molecular and functional characteristics of GABA(B) receptors have been recently extensively studied, the mechanisms underlying receptor desensitization and endocytosis are still poorly understood. We have investigated the effect of continuous agonist exposure on the human GABA(B) receptor functional response and redistribution when expressed in Chinese hamster ovary (CHO-K1) cells. The wild-type GABA(B) receptor-mediated inhibition of the adenylate cyclase activity appeared desensitized after 2 h in the presence of GABA (100 microM). Fusion proteins were generated by attachment of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to GB1 and GB2, respectively, and confocal microscopy experiments in intact living cells semi-stably expressing the constructs were performed. Incubation of co-expressing CFP-GB1 and YFP-GB2 cells in the presence of GABA (100 microM) for 2 h induced a profound receptor internalization, and CFP-GB1 and YFP-GB2 appeared co-localized in the endosome (labelled with Cy3-transferrin). The internalization was blocked by a selective GABA(B) receptor antagonist. These results represent the first clear visualization of agonist-induced internalization of the unique heterodimeric GABA(B) receptor.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. Javier.Maeso@mssm.edu
authors.forename: Javier
authors.initials: J
authors.lastname: González-Maeso
authors.forename: Alan
authors.initials: A
authors.lastname: Wise
authors.forename: Andrew
authors.initials: A
authors.lastname: Green
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Koenig
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects*; D018755:GABA Agonists / Q000494:pharmacology*; D018756:GABA Antagonists / Q000494:pharmacology; D058786:GABA-B Receptor Agonists; D058788:GABA-B Receptor Antagonists; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018080:Receptors, GABA-B / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D001426:Bacterial Proteins; D018755:GABA Agonists; D018756:GABA Antagonists; D058786:GABA-B Receptor Agonists; D058788:GABA-B Receptor Antagonists; D008164:Luminescent Proteins; D021122:Protein Subunits; D018080:Receptors, GABA-B; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D005576:Colforsin; D005680:gamma-Aminobutyric Acid; D000242:Cyclic AMP; D000262:Adenylyl Cyclases