Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the ␣2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits ␣, ␤1, and ␥2 and a number of GPCRs including M4, ␣2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and G␣ proteins shows specificity: there is interaction between the ␣2A receptor and Go, but little interaction between the ␣2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.T wo opposing ideas are invoked to explain how membrane bound signaling proteins transfer information after activation. In the first, components in the membrane freely diffuse and interactions occur through ''collision coupling'' determined by diffusion. Historically, such mechanisms are thought to govern the interaction of G protein-coupled receptor (GPCR) with G protein and the interaction of G protein with downstream enzymes and ion channels. Signal amplification is a key feature of this mechanism (1-3). A second mechanism is the ''physical scaffolding'' hypothesis in which component proteins interact directly or indirectly with each other. The best example of this is the role of InaD in the Drosophila photoreceptor that scaffolds via PDZ domains the light-sensing GPCR rhodopsin, Ca 2ϩ influx TRP channels, phospholipase C, and protein kinase C (4). In principle, this is a way of generating fast activation, fast signal termination, and specificity. A variant of this hypothesis is the localization of proteins in membrane signaling microdomains such as caveolae and lipid rafts.The G protein-gated K ϩ channel (GIRK) was first identified in atrial myocytes. Channel activation occurs after binding of acetylcholine to muscarinic M2 receptors (5) and is responsible for slowing of the heart rate in response to vagal stimulation (6, 7). Analogous GIRK currents are present in neurons and neuroendocrine cells (8). Activation of native and cloned GIRK channels has been shown to involve a direct, membrane-delimited interaction with the G␤␥ subunit (9, 10). One critical point is that the activation occurs rapidly in both native and heterologous settings: complete channel activation can occur within 1 s of the addition of agonist (11-13). Such fast rates of signaling suggest that the components diffuse only small distances, if at all. From these considerations alone it is an appealing hypothesis to propose that the components may be physically scaffolded together. O...

Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the G␣ subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K ؉ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein ␣ subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein ␣ subunit.The G-protein cycle is initiated by binding of an agonist to its target seven-helical G-protein-coupled receptor (GPCR), 1 which associates with a heterotrimeric G-protein on the cytoplasmic side of the cell membrane. Once assembled, this "ternary complex" promotes GDP release and stimulates GTP binding on the G-protein ␣ subunit and dissociation of the G-protein subunits G␣-GTP and G␤␥. Both subunits can activate downstream signaling molecules, including enzymes and ion channels (1, 2). Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. Cloning studies have identified a large RGS gene family, each endowed with a conserved RGS domain of 120 -130 amino acids that is flanked by N and C termini of varying lengths (3-7). By itself, the RGS domain is capable of interacting with G-protein ␣ subunits to accelerate the GTP hydrolysis rate of the G␣ subunit, thereby promoting termination of the G-protein signal (3-7). Based on primary sequence similarities, mammalian RGS proteins have been grouped into five subfamilies (7). In this study we focus largely on RGS8, belonging to the R4 subfamily of RGS proteins that are generally considered prototypical in that they appear to have little function other than to act as GTPase-activating proteins (GAPs) on G i/o and G q/11 G-protein ␣ subunits. However, we also examine RGS7, which belongs to the R7 family, and GAIP, which belongs to the RZ family. RGS7 is particularly interesting because it contains a number of protein-protein interaction domains in the N terminus, but it is of particular relevance for our study because it has a substantially attenuated GAP activity compared with other RG...

Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1؉3.2A) that is activated by the direct binding of G␤␥ subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent G␣ i/o subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A 1 receptor, the adrenergic ␣ 2A receptor, the dopamine D 2S receptor, the M4 muscarinic receptor, and the dimeric GABA-B 1b/2 receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.The activation of G-protein-coupled receptors (GPCRs) 1 by extracellular ligands is an important mechanism involved in a multitude of physiological responses and is of central importance in drug development and therapeutics (1). The activated receptor couples to G-proteins of various subtypes that then activates effector pathways either directly or indirectly. This combination of agonist, receptor, and G-protein is referred to as the "ternary complex" and is thought to be the key essential determinant of the magnitude of the downstream response (2, 3). The most recent formulations propose a cubic ternary complex model with a large number of equilibrium constants between various states governing efficacy (2, 4, 5). The important species is the activated receptor/agonist/G-protein complex. It is proposed that for any combination of these three elements the particular active conformation (or conformational space) is unique and can thus have distinctive signaling consequences (2, 4 -6). An agonist binds more favorably to the active receptor species and thus at equilibrium favors its' formation. Recently this model has been extended to also incorporate the kinetics of G-protein activation and deactivation and indicate that a kinetic model, as opposed to an equilibrium model, may potentially have quite different properties (7).Generally these phenomena have been studied by the use of biochemical assays, using cell homogenates or fractions, or by measuring the behavior of a physiological response many steps downstream from the G-protein cycle. It is apparent that there is a gap in our understanding about how these signaling pathways behave dynamically in intact cells. This is important, because, in reality, the release of hormones and neurotransmitters varies over the second time scale. Agonist binding to receptor is generally agreed to be diffusion-limited and much faster th...

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein ␣ subunits. We have designed G i/o ␣ subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-proteingated inwardly rectifying K ؉ channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G i/o ␣ subunits (G i1 ␣, G i2 ␣, G i3 ␣, and G oA ␣) and a mutation introduced at Cys ؊4 rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G i/o ␣ protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G i/o ␣ subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the G␤␥ dimer by the mutant G␣ subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G i3 ␣ and G oA ␣ ␣ subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.

G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G i/o, in a membrane-delimited fashion by the direct binding of G␤␥ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K ؉ channel, Kir3.1 ؉ 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein ␣ subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein ␣ subunit. With some combinations of agonist͞GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein ␣ subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs.M embers of the family of inwardly rectifying K ϩ (Kir) channels gated by G proteins were first identified in atrial myocytes, where they are activated through stimulation of M 2 muscarinic receptors by acetylcholine (1). Physiologically, activation of this current is partly responsible for slowing of the heart rate in response to vagal-nerve stimulation (2, 3). It is now known that channel activation is membrane-delimited (4), mimicked by nonhydrolyzable GTP analogues (5), and sensitive to pertussis toxin (PTx), implicating the inhibitory family of G proteins (G i/o ) (6). Channel activation occurs because of direct binding of G␤␥ dimers, released from G i/o ␣-containing heterotrimers, to domains on the channel (7-9). G protein-gated Kir channels are also expressed in many central neurones, where they can be activated by a large variety of neurotransmitters acting at G i/o -coupled receptors (10) including ␥-aminobutyric acid (GABA) at the GABA type B (GABA B ) receptor complex and adenosine at A 1 receptors, and they mediate postsynaptic inhibitory events (9,11,12). The molecular counterparts of these currents have now been identified by cloning techniques (13-16): the channel is a heterotetramer of members of the Kir3.0 family of K ϩ channels. Coexpression of Kir3.1 with Kir3.2 or Kir3.4 in heterologous expression systems results in currents that show many of the basic characteristics of the native channels in neurones and atria, respectively.The kinetic behavior of these channels after agonist application and withdrawal has been a subject of intense investigation. To date, these issues have largely been addressed by using...