title: Point mutations in either subunit of the GABAB receptor confer constitutive activity to the heterodimer.
abstract: The GABA receptor (GABABR) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABABR subunit 1 (R1) and GABABR subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABABR mutants as novel tools to explore the molecular mechanisms underlying receptor function. A single amino acid substitution, T290K, in the R1 agonist binding domain results in ligand-independent signaling when this mutant subunit is coexpressed with wild-type R2. Introduction of a Y690V mutation in the putative G protein-coupling domain of R2 is sufficient to confer moderate constitutive activity when this subunit is expressed alone. Activity of the Y690V mutant can be markedly enhanced with coexpression of wild-type R1. Coexpression of both mutant subunits (R1-T290K and R2-Y690K) leads to a further increase in basal signaling. Potencies of the full agonists R-(+)-beta-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABABR variants provided a sensitive probe enabling detection of inverse or partial agonist activity of molecules previously considered neutral antagonists. Our studies using constitutively active isoforms provide independent support for a model of GABABR function that takes into account 1) ligand binding by R1, 2) signal transduction by R2, and 3) modulation of R2-induced function by R1. Furthermore, we demonstrate that certain hallmark features of constitutive activity as originally established with class A GPCRs (e.g., enhanced agonist potency and affinity), are more generally applicable, as suggested by our finding with a class C heterodimeric receptor.
authors.affiliation: Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts-New England Medical Center, Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Richa S
authors.initials: RS
authors.lastname: Mukherjee
authors.forename: Edward W
authors.initials: EW
authors.lastname: McBride
authors.forename: Martin
authors.initials: M
authors.lastname: Beinborn
authors.forename: Kathleen
authors.initials: K
authors.lastname: Dunlap
authors.forename: Alan S
authors.initials: AS
authors.lastname: Kopin
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001418:Baclofen / Q000494:pharmacology*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018755:GABA Agonists / Q000494:pharmacology*; D058788:GABA-B Receptor Antagonists; D006801:Humans; D051379:Mice; D008954:Models, Biological; D015394:Molecular Structure; D017354:Point Mutation; D020033:Protein Isoforms; D011869:Radioligand Assay; D018080:Receptors, GABA-B / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D018755:GABA Agonists; D058788:GABA-B Receptor Antagonists; D020033:Protein Isoforms; D018080:Receptors, GABA-B; D001418:Baclofen