Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling

The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.     

RGS2 interacts with Gs and adenylyl cyclase in living cells

Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.     

ERK activation by G-protein-coupled receptors in mouse brain is receptor identity-specific.

In transfected cells and non-neuronal tissues many G-protein-coupled receptors activate p44/42 MAP kinase (ERK), a kinase involved in both hippocampal synaptic plasticity and learning and memory. However, it is not clear to what degree these receptors couple to ERK in brain. G(s)-coupled beta-adrenergic receptor activation of ERK in neurons is critical in the regulation of synaptic plasticity in area CA1 of the hippocampus. In addition, alpha(1)- and alpha(2)-adrenergic receptors, present in CA1, could potentially activate ERK. We find that, like the beta-adrenergic receptor, the G(q)-coupled alpha(1)AR activates ERK in adult mouse CA1. However, activation of the G(i/o)-coupled alpha(2)AR does not activate ERK, nor does activation of a homologous G(i/o)-coupled receptor enriched in adult mouse CA1, the 5HT(1A) receptor. In contrast, the nonhomologous G(i/o)-coupled gamma-aminobutyric acid type B receptor does activate ERK in adult mouse CA1. Surprisingly, activation of alpha(2)ARs in CA1 from immature animals where basal phospho-ERK is low induces ERK phosphorylation. These data suggest that although most G-protein-coupled receptor subtypes activate ERK in non-neuronal cells, the coupling of G(i/o) to ERK is tightly regulated in brain.     

Dexras1/AGS-1 inhibits signal transduction from the Gi-coupled formyl peptide receptor to Erk-1/2 MAP kinases.

Dexras1 is a novel GTP-binding protein (G protein) that was recently discovered on the basis of rapid mRNA up-regulation by glucocorticoids in murine AtT-20 corticotroph cells and in several primary tissues. The human homologue of Dexras1, termed activator of G protein signaling-1 (AGS-1), has been reported to stimulate signaling by G(i) heterotrimeric G proteins independently of receptor activation. The effects of Dexras1/AGS-1 on receptor-initiated signaling by G(i) have not been examined. Here we report that Dexras1 inhibits ligand-dependent signaling by the G(i)-coupled N-formyl peptide receptor (FPR). Dexras1 and FPR were transiently co-expressed in both COS-7 and HEK-293 cells. Activation of FPR by ligand (N-formyl-methionine-leucine-phenylalanine (f-MLF)) caused phosphorylation of endogenous Erk-1/2 that was reduced by co-expression of Dexras1. Direct effects of Dexras1 on the activity of co-expressed, epitope-tagged Erk-2 (hemagglutinin (HA)-Erk-2) were measured by immune complex in vitro kinase assay. Expression of Dexras1 alone resulted in a 1.9- to 4.9-fold increase in HA-Erk-2 activity; expression of the unliganded FPR alone resulted in a 6.2- to 8.1-fold increase in HA-Erk-2 activity. Stimulation of FPR by f-MLF produced a further 8- to 10-fold increase in HA-Erk-2 activity over the basal (non-ligand-stimulated) state, and this ligand-dependent activity was attenuated at the time points of maximal activity by co-expression of Dexras1 (reduced 31 +/- 6.8% in COS-7 at 10 min and 86 +/- 9.2% in HEK-293 at 5 min, p < 0.01 for each). Expression of Dexras1 did not influence protein expression of FPR or Erk, suggesting that the inhibitory effects of Dexras1 reflect a functional alteration in the signaling cascade from FPR to Erk. Expression of Dexras1 had no effect on expression of G(i)alpha species, but significantly impaired pertussis toxin-catalyzed ADP-ribosylation of membrane-associated G(i)alpha. Expression of Dexras1 also significantly decreased in vitro binding of GTPgammaS in f-MLF-stimulated membranes of cells co-transfected with FPR. These data suggest that Dexras1 inhibits signal transduction from FPR to Erk-1/2 through an effect that is very proximal to receptor-G(i) coupling. While Dexras1 weakly activates Erk in the resting state, more potent effects are evident in the modulation of ligand-stimulated receptor signal transduction, where Dexras1 functions as an inhibitor rather than activator of the Erk mitogen-activated protein kinase signaling cascade.     

Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

The olfactory G protein Gαolf possesses a lower GDP-affinity and deactivates more rapidly than Gsαshort: consequences for receptor-coupling and adenylyl cyclase activation

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.     

Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules

Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the mu-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G(beta) subunit of G(beta gamma). The aggregate effect of these changes is to shift mu-opioid receptor-coupled signaling from predominantly G(i alpha) inhibitory to (G(i)-derived) G(beta gamma) stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G(s), which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.     

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.     

FRET-based monitoring of conformational change of the beta2 adrenergic receptor in living cells

The beta(2) adrenergic receptor (beta(2)AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of beta(2)AR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant beta(2)ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of beta(2)AR in living cells was more rapid than that of purified beta(2)AR measured in vitro. Interestingly, the direction of the emission ratio change of beta(2)AR was opposite to that of the norepinephrine-responsive alpha(2A) adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase.     

Co-expression of the beta2-adrenoceptor and dopamine D1-receptor with Gsalpha proteins in Sf9 insect cells: limitations in comparison with fusion proteins

The G-protein G(salpha) exists in three isoforms, the G(salpha) splice variants G(salphashort) (G(salphaS)) and G(salphalong) (G(salphaL)), and the G-protein G(alphaolf) that is not only involved in olfactory signaling but also in extrapyramidal motor regulation. Studies with beta(2)-adrenoceptor (beta(2)AR)-G(salpha) fusion proteins showed that G(salpha) proteins activate adenylyl cyclase (AC) in the order of efficacy G(salphaS)>G(salphaL) approximately G(alphaolf) and that G(salpha) proteins confer the hallmarks of constitutive activity to the beta(2)AR in the order of efficacy G(salphaL)>G(alphaolf)>G(salphaS). However, it is unclear whether such differences between G(salpha) proteins also exist in the nonfused state. In the present study, we co-expressed the beta(2)AR and dopamine D(1)-receptor (D(1)R) with G(salpha) proteins at different ratios in Sf9 insect cells. In agreement with the fusion protein studies, nonfused G(alphaolf) was less efficient than nonfused G(salphaS) and G(salphaL) at activating AC, but otherwise, we did not observe differences between the three G(salpha) isoforms. Thus, it is much easier to dissect differences between G(salpha) isoforms using beta(2)AR-G(salpha) fusion proteins than nonfused G(salpha) isoforms.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

AMP-Activated protein kinase is activated by the stimulations of G(q)-coupled receptors

The AMP-activated protein kinase (AMPK) functions as a metabolic sensor that monitors cellular AMP and ATP levels. Platelet-activating factor (PAF) activates endogeneous AMPKalpha1 in Chinese hamster ovary cells expressing the PAF receptor coupled with both G(i) and G(q), but its activity was not inhibited after treatment with islet-activating protein. Norepinephrine and bradykinin also activated AMPKalpha1 in cells expressing the G(q)-coupled alpha(1b)-adrenergic receptor and bradykinin receptor, respectively. Stimulations of the G(i)-coupled alpha(2A)-adrenergic receptor, fMet-Leu-Phe receptor, prostaglandin EP3alpha receptor, and G(s)-coupled beta(2)-adrenergic receptor did not activate AMPKalpha1. AMPKalpha1 thus is activated specifically by stimulation of G(q)-coupled receptors. G(q)-coupled receptors transmit the signal for GLUT4 translocation and glucose uptake through an insulin-independent pathway. However, direct activation of AMPKalpha1 with treatment of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside did not trigger GLUT4 translocation nor stimulate glucose uptake in our cells. Thus, activation of AMPKalpha1 via G(q) is not sufficient to trigger GLUT4 translocation or stimulate glucose uptake.     

Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.     

Gs activation is time-limiting in initiating receptor-mediated signaling

To analyze individual steps of G(S)-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G(S) activation in living cells. This was done by coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gbetagamma subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G(S) signaling cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction was on the same time scale as A(2A) receptor activation with a time constant of <50 ms. G(S) activation was markedly slower and around 450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors. Taken together, our kinetic measurements demonstrate that the rate of G(S) activation limits initiation of G(S)-coupled receptor signaling.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.     

A highly effective dominant negative alpha s construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative alpha(s) mutant containing substitutions that alter distinct functions was produced and tested for its effects on G(s)-coupled receptor activity in HEK-293 cells. Mutations in the alpha3beta5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for betagamma, and A366S, which decreases affinity for GDP. This triple alpha(s) mutant can inhibit signaling to G(s) from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this alpha(s) mutant blocks all signaling from the calcitonin receptor to G(q). These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein alpha subunits can compete with each other for binding to the same receptor. This dominant negative alpha(s) construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

Discrete amino acid sequences of the alpha 1-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis.

We have constructed a variety of chimeric beta 2/alpha 1 adrenergic receptors (AR) in which selected portions of the third intracellular loop of the alpha (1B)AR were substituted into the corresponding regions of the beta 2AR. The mutant receptors were both transiently and permanently expressed in COS-7 or L-cells, respectively, and tested for their ability to mediate epinephrine-induced activation of polyphosphoinositide (PI) hydrolysis and adenylylcyclase. We have determined that 27 amino acids of the alpha (1B)AR (residues 233-259) derived from the N-terminal portion of the third intracellular loop represent the structural determinant conferring to the beta 2AR the ability to activate PI hydrolysis. This finding suggests that in the alpha (1B)AR the N-terminal portion of the third intracellular loop plays a major role in determining the selectivity of receptor-G protein coupling. However, replacement of alpha 1B sequences in the third intracellular loop of the beta 2AR did not abolish the latter receptor's coupling to activation of adenylylcyclase, thus resulting in chimeric adrenergic receptors which activated both PI hydrolysis and adenylylcyclase. These results indicate that, even if the N-terminal portion of the third intracellular loop is a major determinant of the selectivity of receptor-G protein coupling, other structural domains of the receptors also modulate this property. The comparison of the amino acid sequences which determine the selectivity of G protein coupling in functionally similar receptors may help to elucidate the structural basis for activation of specific G protein-effector systems.