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Point Mutations in Either Subunit of the GABA_B Receptor Confer Constitutive Activity to the Heterodimer

Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts (R.S.M., E.W.M., M.B., A.S.K.); and Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts (A.S.K., K.D.)

The GABA receptor (GABA_BR) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABA_BR subunit 1 (R1) and GABA_BR subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABA_BR 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-(+)-β-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABA_BR 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 GABA_BR 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.

Address correspondence to: Dr. Alan S. Kopin, 750 Washington St., P.O. Box 7703, Boston, MA 02111. E-mail: akopin{at}tufts-nemc.org

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