TY - JOUR T1 - Point Mutations in the Transmembrane Region of GABA<sub>B2</sub> Facilitate Activation by the Positive Modulator <em>N,N</em>′-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) in the Absence of the GABA<sub>B1</sub> Subunit JF - Molecular Pharmacology JO - Mol Pharmacol SP - 2027 LP - 2036 DO - 10.1124/mol.106.028183 VL - 70 IS - 6 AU - Delphine S. Dupuis AU - Dinko Relkovic AU - Loic Lhuillier AU - Johannes Mosbacher AU - Klemens Kaupmann Y1 - 2006/12/01 UR - http://molpharm.aspetjournals.org/content/70/6/2027.abstract N2 - GABAB receptors are heterodimers of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Agonists such as GABA and baclofen bind to the GB1 subunit only, whereas GB2 is essential for G protein activation. Positive allosteric modulators enhance the potency and efficacy of agonists at GABAB receptors and are of particular interest because they lack the sedative and muscle relaxant properties of agonists. In this study, we aimed to characterize the interaction of the positive modulator N,N′-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABAB receptor heterodimer. Using functional guanosine 5′-O-(3-[35S]thio)triphosphate binding assays, we observed positive modulation by GS39783 in different vertebrate species but not in Drosophila melanogaster. However, coexpression of D. melanogaster GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/D. melanogaster GB2 subunit chimeras, this pointed to a critical role of the GB2 transmembrane region for positive modulation. We further characterized GS39783 function using point mutations. GS39783 positively modulated GABA responses but also showed considerable agonistic activity at heterodimers containing a mutant rat GB2 subunit with three amino acid substitutions in transmembrane domain VI. It was surprising that in contrast to wild-type rat GB2, this mutant subunit was also activated by GS39783 when expressed without GB1. The mutations of both G706T and A708P are necessary and sufficient for activation and identify a key region for the effect of GS39783 in the GB2 transmembrane region. Our data show that mutations of specific amino acids in GB2 can induce agonism in addition to positive modulation and facilitate GB2 activation in the absence of GB1. The American Society for Pharmacology and Experimental Therapeutics ER -