These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein ␣ subunit function. However, using a peptide that binds G␤␥ (␤ARKct), we show that G␤␥ is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the ␤ 2 -adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, ␤ 2 AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

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...

Bioluminescence resonance energy transfer (BRET)and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several Gprotein subunits (G␣ ␣ s , G␣ ␣ i , G␤ ␤ 1 and G␥ ␥ 2 ) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically G␤ ␤ 1 -YFP 1-158 and G␥ ␥ 2 -YFP 159-238 , which heterodimerize to produce fluorescent YFP-G␤ ␤ 1 ␥ ␥ 2 ). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-G␥ ␥ 2 , GFP-G␤ ␤ 1 or YFP-G␤ ␤ 1 ␥ ␥ 2 . G␣ ␣ subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and G␤ ␤␥ ␥ was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.