Identification of a GABAB Receptor Subunit, gb2, Required for Functional GABAB Receptor Activity

Abstract

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant ␥-aminobutyric acid, type B (GABA B ) receptor (gb1a) and a GABA B -like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K ؉ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABA B receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABA B receptor.Metabotropic GABA B 1 receptors were first distinguished pharmacologically by Hill and Bowery (1). Kaupmann et al. (2) recently cloned two alternatively spliced forms of the GABA B receptor, termed gb1a and gb1b, which belong to the G proteincoupled receptor superfamily and are most closely related to the metabotropic glutamate receptors. Although native GABA B receptors are reported to activate inwardly rectifying K ϩ channels (Kir) (3), recombinant gb1a receptors coexpressed with Kir channels in Xenopus oocytes failed to be functionally active (2,4,12). A recent report has shown that recombinant GABA B receptors fail to couple to effector pathways in a variety of non-neuronal and neuronal cell types, suggesting that additional cellular component(s) are required (4). Failure to show GABA B receptor function coupled with previous reports that GPCRs can undergo dimerization (5-8), suggested that heterodimerization may be important for GABA B receptor function. Furthermore, receptor heterodimerization appears to rescue function of mutated or chimeric muscarinic and adrenergic receptors (9). We report here that coexpression of gb1 and gb2 receptors are necessary for the formation of functional GABA B receptors and result in heterodimerization. EXPERIMENTAL PROCEDURESExpression Constructs for gb1a and gb2 Receptors-The murine gb1a (mgb1a) cDNA was constructed from two expressed sequence tags (IMAGE Consortium clone identification numbers 472408 and 319196), combined by standard PCR methods, and subcloned into the pCINeo (Stratagene) and pcDNA3.1 (Invitrogen) vectors.2 The rat gb1a receptor was obtained by PCR of rat brain cDNA using oligonucleotide primers based on the published sequence (Ref. 2; GenBank TM accession number Y10369) and subcloned into pcDNA3.1.Two independently der...

The GABA B receptor interacts directly with the related transcription factors CREB2 and ATFx

“…However, when expressed recombinantly, GABA B -R1 failed to reproduce the expected agonist affinities (3,5) and was not expressed at the cell surface (5,9), suggesting that a component was lacking from GABA B -R1 for reconstitution of the functional receptor. Subsequent studies confirmed that a second distinct but related GPCR, GABA B -R2, heterodimerizes with GABA B -R1 to create the GABA B -receptor (4)(5)(6)(7)(8). Yeast two-hybrid (YTH) studies showed that the two receptors interact through a coiled-coil domain within their respective C-terminal tails (5,7,10), and in situ hybridization analysis showed colocalization of the two receptors (4,5,7,10).…”

“…GABA B receptors were originally identified pharmacologically (2) and couple through G proteins to Ca 2ϩ or K ϩ channels. Despite being recognized many years ago, the molecular nature of the GABA B receptor has been elucidated only recently (3)(4)(5)(6)(7)(8). The initial GABA B ''receptor,'' GABA B -R1, was expression cloned by using a high-affinity antagonist and showed homology to Family C G protein-coupled receptors (GPCRs), such as metabotropic glutamate receptors, with a characteristically large extracellular N-terminal domain as well as a seven-transmembrane topology (3).…”

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

“…For example, transmembrane domain peptides that inhibit oligomerization of ␤ 2 -adrenergic receptors can attenuate signal transduction in vitro (10), and oligomerization-defective mutants of a yeast GPCR can bind agonist normally but are signaling impaired (11). Strikingly, heterodimerization between the R1 and R2 subunits of the GABA(B) receptor is essential for signal transduction because the R1 subunit can bind agonist but cannot activate G proteins, whereas the R2 subunit cannot bind agonist but can activate G proteins (5,(12)(13)(14)(15).…”

Heterodimeric coiled-coil interactions of human GABA B receptor

“…Formation of a functional GABA B receptor requires the heterodimeric assembly of GABA B receptor 1 (GBR1) and GABA B receptor 2 (GBR2) subunits (3)(4)(5)(6)(7). Both consist of an N-terminal extracellular domain, a seven-helix transmembrane domain, and a C-terminal intracellular domain.…”