Reconstitution of catecholamine-stimulated guanosine triphosphatase activity

Brandt, Douglas R.; Asano, Taku; Pedersen, Steen E.; Ross, Elliott M.

doi:10.1021/bi00288a002

Cited by 214 publications

(91 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Reconstitution of P2Y 1 -R into Proteoliposomes. Purified P2Y 1 -R was co-reconstituted with either G␣ q or G␣ 11 and G␤ 1 ␥ 2 into proteoliposomes by a modification of the method used by Brandt et al (1983) and as described in detail by Cunningham et al (2001). Briefly, 15 pmol of purified P2Y 1 -R, 50 pmol of G␣, and 150 pmol of G␤ 1 ␥ 2 were reconstituted by Sephadex G-50 chromatography into phospholipid vesicles containing L-␣-phosphatidylserine from brain, L-␣-phosphatidylethanolamine from liver, and cholesteryl hemisuccinate.…”

Section: Methodsmentioning

confidence: 99%

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Waldo

Harden²

2004

Mol Pharmacol

118

View full text Add to dashboard Cite

The human P2Y 1 receptor (P2Y 1 -R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y 1 -R antagonist [ binding assays for antagonists with the purified P2Y 1 -R were in good agreement with the K i and K B values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y 1 -R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) Ͼ ADP ϭ 2-methylthio-ATP ϭ adenosine-5Ј-O-(3-thio)triphosphate ϭ adenosine-5Ј-O-(2-thiodiphosphate) Ͼ Ͼ ATP was obtained. The signaling activity of the purified P2Y 1 -R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y 1 -R and G␣ q ␤ 1 ␥ 2 was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC 50 values of agonists for activation of the purified P2Y 1 -R were similar to their respective K i values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC 50 and K i values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-␤1 were potent and efficacious GTPase-activating proteins for G␣ q and G␣ 11 in P2Y 1 -R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y 1 -R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.Burnstock's hypothesis (Burnstock, 1972) of purinergic signaling largely has been confirmed over the past two decades by demonstration of regulated release of cellular nucleotides, by elucidation of the mechanisms of metabolism of extracellular nucleotides by a complex array of ecto-enzymes, and by delineation of myriad nucleotide-promoted physiological responses (Dubyak and El-Moatassim, 1993;Harden et al., 1995;Ralevic and Burnstock, 1998;Lazarowski et al., 2003). Moreover, the molecular cloning of two classes of ubiquitously distributed receptors for extracellular nucleotides, the ligand-gated P2X receptors (Khakh et al., 2000) and the G protein-coupled P2Y receptors , comprising at least 15 human genes, has amplified the importance of nucleotide-promoted signaling.Although a burgeoning interest in the biology and therapeutic potential of extracellular nucleotide signaling now exists, the complex regulatory pathways that underlie the action of extracellular nucleotides remain difficult to study. Few truly selective agonists and antagonists are available for the P2 receptors, and studies with intact tissues are compromised both by nucleotide release and by metabolism and interconversion of ...

show abstract

Section: Methodsmentioning

confidence: 99%

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Waldo

Harden²

2004

Mol Pharmacol

118

View full text Add to dashboard Cite

show abstract

“…Receptor activation increases the rate of GDP dissociation without affecting the intrinsic rate of GTPase and thus leads to the exchange of GTP for GDP in the presence of GTP (Higashijima et al, 1987). This is observed as an increased rate of steady-state GTPase (Brandt et al, 1983;Cerione et al, 1984), which is correlated with the efficacy of the ligand used to stimulate the receptor (Costa and Hertz, 1989). 4) Inhibition of the GTPase activity by CTX-mediated ADP ribosylation of G␣ (Johnson et al, 1978) or by mutations in the G␣ subunit increases the constitutive activity of the G protein in the presence of GTP, whereas mutations or regulating factors, such as regulators of G protein signaling, that increase the GTPase activity inhibit G protein activation (Watson et al, 1996).…”

Section: Guanine Nucleotide Exchange-independent Activation Of G S 725mentioning

confidence: 99%

Guanine Nucleotide Exchange-Independent Activation of G_sProtein by β₂-Adrenoceptor

Uğur

Öner²,

Molinari³

et al. 2005

Mol Pharmacol

View full text Add to dashboard Cite

␤ 2 -Adrenoceptor-mediated activation of G s and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the ␣-subunit of the G protein.Here we showed that a ␤ 2 -adrenoceptor-G s system, heterologously expressed in cyc Ϫ or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5Ј-O-(2-thiodiphosphate) (GDP␤S). The potency and maximal ability of GDP to activate G s and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentrationdependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP (ϳ5% GDP) from GDP. Moreover, the activation of G s in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate ␤-adrenoceptor-induced activation of G s -adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for ␤ 2 -adrenoceptor-G sadenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptorinduced G s protein activation.Heterotrimeric G proteins and their cognate heptahelical membrane receptors constitute the largest family of transmembrane signaling proteins. According to the accepted model of G protein activation, the GTP-bound form of G protein is active, whereas the GDP-bound form is inactive and the receptor-mediated exchange of GDP with GTP on the nucleotide-binding site is necessary and sufficient to initiate the activation process. In turn, deactivation is achieved by the hydrolysis of bound GTP to GDP. Fine-tuning of the rates of the elementary steps involved in this activation-deactivation cycle determines the average activity of the G protein (i.e., the amount of accumulated G␣-GTP), thus the strength of the signal transmitted to the inside of the cell (for review see Gilman, 1987;Hamm, 1998). This scenario implies that GTP is the natural activator of the G proteins, whereas GDP is their universal inhibitor, and that GTPase reaction is the off-switch for the G protein-mediated signal transduction. Therefore, the presence of excess GDP is expected to inhibit receptor-induced G protein activation, at least partly, by competing with GTP and the potency of this inhibitory effect should be correlated with the ability of the receptor's ligand to displace GDP from G␣. Viewed from a more rigorous standpoint, the sensitivity o...

show abstract

“…GTPase Assays-Purified recombinant human P2Y 1 receptor 2 was reconstituted with G␣ q and G␤ 1 ␥ 2 into proteoliposomes by a modification of the method described by Brandt et al (21). Briefly, 15 pmol of P2Y1 receptor, 40 pmol of G␣ q , and 150 pmol of G␤ 1 ␥ 2 were combined with a mixture of phosphatidylethanolamine, phosphatidylserine, and cholesterol hemisuccinate in detergent solution.…”

mentioning

confidence: 99%

Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling

Cunningham

Waldo

Hollinger

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate G␣ q -mediated inositol lipid signaling. Although purified RGS2 blocks PLC-␤ activation by the nonhydrolyzable GTP analog guanosine 5-O-thiophosphate (GTP␥S), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTP␥S-stimulated G␣ 11 signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10 -20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-␤t activity than of GTP␥S-promoted PLC-␤t activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4␤-phorbol 12␤-myristate 13␣-acetate and, to a lesser extent, by the P2Y 2 receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTP␥S-stimulated PLC-␤t activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y 1 receptor and G␣ q ␤␥. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.A variety of hormone and neurotransmitter receptors transduce signals through heterotrimeric G proteins. In their inactive state, G proteins exist as heterotrimers consisting of ␣, ␤, and ␥ subunits with GDP bound to G␣. Upon agonist occupation, the receptor promotes GDP/GTP exchange, and the active GTP-bound G␣ subunit and G␤␥ dissociate to interact with target effector proteins. Signaling is terminated by the hydrolysis of GTP to GDP and the subsequent formation of the heterotrimer. Therefore, the magnitude and duration of signaling is determined by the length of time G␣ remains in the active GTP-bound conformation.A recently identified family of proteins termed RGS (regulators of G protein signaling) proteins interact directly with G␣ subunits to decrease the lifetime of the active GTP-bound complex (1-4). RGS proteins attenuate heterotrimeric G protein 1 signaling by functioning as both GTPase-activating proteins (GAPs) (5, 6) and inhibitors of G protein/effector interaction (6, 7). In vitro studies illustrate that RGS2 interacts with and fu...

show abstract

Reconstitution of catecholamine-stimulated guanosine triphosphatase activity

Cited by 214 publications

References 21 publications

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Guanine Nucleotide Exchange-Independent Activation of G_sProtein by β₂-Adrenoceptor

Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling

Contact Info

Product

Resources

About

Reconstitution of catecholamine-stimulated guanosine triphosphatase activity

Cited by 214 publications

References 21 publications

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y1Receptor

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y1Receptor

Guanine Nucleotide Exchange-Independent Activation of GsProtein by β2-Adrenoceptor

Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling

Contact Info

Product

Resources

About

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Guanine Nucleotide Exchange-Independent Activation of G_sProtein by β₂-Adrenoceptor