The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.
The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited G␣ s -Q227L-or  2 -adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C 1 but not the C 2 domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by G␣ i . These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.Heterotrimeric G protein-mediated receptor signaling pathways are pivotal parts of the intricate and diverse biological processes dictating cellular function. G proteins transduce signals to a variety of effectors including adenylyl cyclase (AC) 1 (1). The hormone-sensitive AC system is a typical archetype of G protein-mediated signal transduction. Appropriate agonistbound, heptahelical receptors activate G s by catalyzing the exchange of GDP for GTP. The GTP-bound ␣ subunit of G s in turn activates AC, increasing the rate of synthesis of cyclic AMP from ATP (1, 2). All isoforms of mammalian AC are stimulated by the heterotrimeric G protein G s . Many other regulatory influences including RGS (regulators of G protein signaling) proteins have crucial effects on various AC isoforms (3). These enzymes thus serve critical roles as integrators of diverse inputs.RGS2 is a 211-amino acid protein that like other members of this family mediates its GAP activity via its core domain (4 -6). Experiments using recombinant RGS2 and membranes prepared from insect cells (Sf9) expressing different AC isoforms indicate that RGS2 suppresses the activity of AC III, the predominant isotype in the olfactory system, and the cardiac isoforms, V and VI (3). RGS2 has also been shown to decrease cAMP accumulation in TC3 insulinoma cells (7). When added to purified recombinant type V AC cytoplasmic domains, recombinant RGS2 decreases cAMP production stimulated by either G␣ s or forskolin. The structural basis for the inhibitory effect of RGS2 remains unknown, although it is likely a direct effect. In vivo, odorant-elicited cAMP stimulation resu...
Many Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTPase activity of G(ialpha) and G(qalpha)-subunits [i.e., behave as GTPase-activating proteins (GAPs)] and several act as G(qalpha)-effector antagonists. RGS3, a structurally distinct RGS member with a unique N-terminal domain and a C-terminal RGS domain, and an N-terminally truncated version of RGS3 (RGS3CT) both stimulated the GTPase activity of G(ialpha) (except G(zalpha)) and G(qalpha) but not that of G(salpha) or G(12alpha). RGS3 and RGS3CT had G(qalpha) GAP activity similar to that of RGS4. RGS3 impaired signaling through G(q)-linked receptors, although RGS3CT invariably inhibited better than did full-length RGS3. RGS3 potently inhibited G(qalpha)Q209L- and G(11alpha)Q209L-mediated activation of a cAMP-response element-binding protein reporter gene and G(qalpha)Q209L induced inositol phosphate production, suggesting that RGS3 efficiently blocks G(qalpha) from activating its downstream effector phospholipase C-beta. Whereas RGS2 and to a lesser extent RGS10 also inhibited signaling by these GTPase-deficient G proteins, other RGS proteins including RGS4 did not. Mutation of residues in RGS3 similar to those required for RGS4 G(ialpha) GAP activity, as well as several residues N terminal to its RGS domain impaired RGS3 function. A greater percentage of RGS3CT localized at the cell membrane than the full-length version, potentially explaining why RGS3CT blocked signaling better than did full-length RGS3. Thus, RGS3 can impair Gi- (but not Gz-) and Gq-mediated signaling in hematopoietic and other cell types by acting as a GAP for G(ialpha) and G(qalpha) subfamily members and as a potent G(qalpha) subfamily effector antagonist.
Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Gα α α α subunits, Gα α α α i has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.
G 12 ␣/G 13 ␣ transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G 12 ␣/ G 13 ␣ signaling to an SRE reporter gene.
Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.
Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which G␣ i and G␣ q hydrolyze GTP to GDP, thereby limiting the duration in which GTP-G␣ i and GTP-G␣ q can activate effectors. Since GDP-G␣ subunits rapidly combine with free G␥ subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of G␥ subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind G 5 in the absence of a G␥ subunit, RGS proteins are not known to directly influence G␥ signaling. Here we show that RGS3 binds G 1 ␥ 2 subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with G 1 ␥ 2 inhibits G 1 ␥ 2 -induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of G 1 ␥ 2 signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect G 1 ␥ 2 signaling in these assays. Consistent with the in vivo results, RGS3 inhibits G␥-mediated activation of phospholipase C in vitro. Thus, RGS3 may limit G␥ signaling not only by virtue of its GTPase-activating protein activity for G␣ subunits, but also by directly interfering with the activation of effectors.Heterotrimeric G-proteins link seven transmembrane receptors to downstream signaling pathways. Receptor activation triggers the exchange of GTP for GDP by the G␣ subunit of the heterotrimeric G-protein, causing a conformational change in the G␣ subunit, which facilitates its dissociation from the receptor and G␥ subunits. GTP-bound G␣ and free G␥ subunits then bind and activate downstream effectors. However, G␣ subunits possess an intrinsic GTPase activity, which returns G␣ to its GDP bound state and thereby limits the duration of G␣ signaling. Because GDP-G␣ possesses a high affinity for G␥ subunits, the heterotrimeric G-protein rapidly reforms, effectively ending G␥-mediated signaling as well (reviewed in Refs. 1 and 2).Cells possess another important mechanism that curtails the duration in which a G␣ subunit remains GTP bound. Members of a family of proteins termed regulators of G-protein signaling (RGS) 1 dramatically accelerate the intrinsic rate that certain G␣ subunits hydrolyze GTP, a property that identifies them as GTPase-activating proteins (GAPs). The mammalian RGS proteins have a 120-amino acid region, RGS domain, or RGS box, which binds G␣ i and G␣ q subfamily members in a transition state of the GTP hydrolysis reaction, thereby lowering the free energy of the reaction (reviewed in Refs. 3 and 4). In addition, Rho guanine nucleotide exchange factors have a divergent RGS domain, which accelerates the intrinsic GTPase activity of G␣ 12 and G␣ 13 (5, 6). RGS proteins with GAP activity for members of the G␣ s subfamily remain enigmatic.The mammalian RGS proteins ca...
Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.
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