2'(3')-O-(N-methylanthraniloyl)-substituted GTP analogs: a novel class of potent competitive adenylyl cyclase inhibitors

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.     

The signal transfer regions of G alpha(s)

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.     

Fluorescence analysis of receptor-G protein interactions in cell membranes

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.     

Receptor-independent activators of heterotrimeric G-protein signaling pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.     

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.     

Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties

In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.     

G(alpha)(i) controls the gating of the G protein-activated K(+) channel, GIRK.

GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.     

Activation-induced subcellular redistribution of G alpha(s) is dependent upon its unique N-terminus

The heterotrimeric G protein subunit, alpha(s), can move reversibly from plasma membranes to cytoplasm in response to activation by GPCRs or activating mutations. We examined the importance of the unique N-terminus of alpha(s) in this translocation in cultured cells. alpha(s) contains a single site for palmitoylation in its N-terminus, and this was replaced by different plasma membrane targeting motifs. These N-terminal alpha(s) mutants were targeted properly to plasma membranes, capable of coupling activated GPCRs to effectors, and able to constitutively stimulate cAMP production when they also contained an activating mutation. However, when activated by a constitutively activating mutation or by agonist-activated beta-AR, these N-terminal alpha(s) mutants failed, for the most part, to undergo redistribution from plasma membranes to cytoplasm, as assayed by immunofluorescence microscopy, or from a particulate to soluble fraction, as assayed by subcellular fractionation. These results highlight the importance of the extreme N-terminus of alpha(s) and its single site of palmitoylation for facilitating activation-induced translocation and provide insight into the mechanism of this G protein trafficking event.     

G alpha i-G alpha s chimeras define the function of alpha chain domains in control of G protein activation and beta gamma subunit complex interactions

Gs and Gi2 are G proteins whose alpha subunits are 65% homologous. Within the 355 amino acid alpha i2 polypeptide, substitution of residues Ile213-Lys319 with the corresponding alpha s region (Ile235-Arg356) generated a chimera that activated adenylyl cyclase, indicating that the alpha s activation domain resides within this 122 amino acid alpha s sequence. Mutation within alpha s residues Glu15-Pro144 resulted in an alpha s polypeptide having an enhanced rate of GDP dissociation. Mutation within two regions of the N-terminus influenced the ability of pertussis toxin to ADP-ribosylate the alpha subunit polypeptide, a reaction controlled by the beta gamma subunit complex. The findings define the G protein alpha subunit N-terminus as a regulatory region controlling beta gamma subunit interactions and GDP dissociation independent of the GTPase and effector activation domains.     

Conditional stimulation of type V and VI adenylyl cyclases by G protein betagamma subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.     

Basic fibroblast growth factor utilizes both types of component subunits of Gs for dual signaling in human adipocytes. Stimulation of adenylyl cyclase via Galph(s) and inhibition of NADPH oxidase by Gbeta gamma(s)

Basic fibroblast growth factor (bFGF), a ligand of receptor protein-tyrosine kinases, promoted the dissociation of G(s) and had antagonistic stimulatory and inhibitory effects on adenylyl cyclase and NADPH oxidase in human fat cell plasma membranes. The bFGF-induced activation of adenylyl cyclase was blocked by COOH-terminal anti-Galpha(s), indicating that it was mediated by Galpha(s). The inhibitory action of bFGF was mimicked by exogenously supplied Gbetagamma-subunits and was reversed by anti-Gbeta(1/2), or betaARK-CT, a COOH-terminal beta-adrenergic receptor kinase fragment that specifically binds free Gbetagamma, indicating that it was transduced by Gbetagamma complexes. The bFGF-induced inhibition of NADPH-dependent H(2)O(2) generation was also reversed by peptide 100-119, an inhibitor of G(s) activation by ligand-occupied beta-adrenergic receptors, indicating that the Gbetagamma complexes mediating the inhibitory action of the growth factor are derived from G(s). The findings suggest a direct, non-kinase-dependent, coupling of bFGF receptor(s) to G(s) and provide the first example of a ligand of receptor protein-tyrosine kinases that is capable of utilizing both types of component subunits of a single heterotrimeric G protein for dual signaling in a single cell type.     

Gbetagamma subunit combinations differentially modulate receptor and effector coupling in vivo

In vitro, little specificity is seen for modulation of effectors by different combinations of Gbetagamma subunits from heterotrimeric G proteins. Here, we demonstrate that the coupling of specific combinations of Gbetagamma subunits to different receptors leads to a differential ability to modulate effectors in vivo. We have shown that the beta(1)AR and beta(2)AR can activate homomultimers of the human inwardly rectifying potassium channel Kir 3.2 when coexpressed in Xenopus oocytes, and that this requires a functional mammalian Gs heterotrimer. Modulation was independent of cAMP production, suggesting a membrane-delimited mechanism. To analyze further the importance of different Gbetagamma combinations, we have tested the facilitation of Kir 3.2 activation by betaAR mediated by different Gbetagamma subunits. The subunits tested were Gbeta(1,5) and Ggamma(1,2,7,11). These experiments demonstrated significant variation between the ability of the Gbetagamma combinations to activate the channels after receptor stimulation. This was in marked contrast to the situation in vitro where little specificity for binding of a Kir 3.1 C-terminal GST fusion protein by different Gbetagamma combinations was detected. More importantly, neither receptor, although homologous both structurally and functionally, shared the same preference for Gbetagamma subunits. In the presence of beta(1)AR, Gbeta(5)gamma(1) and Gbeta(5)gamma(11) activated Kir 3.2 to the greatest extent, while for the beta(2)AR, Gbeta(1)gamma(7), Gbeta(1)gamma(11,) and Gbeta(5)gamma(2) produced the greatest responses. Interestingly, no preference was seen in the ability of different Gbetagamma subunits to facilitate receptor-stimulated GTPase activity of the Gsalpha. These results suggest that it is not the receptor/G protein alpha subunit interaction or the Gbetagamma/effector interaction that is altered by Gbetagamma, but rather that the ability of the receptor to interact productively with the Gbetagamma subunit directly and/or the G protein/effector complex is dependent on the specific G protein heterotrimer associated with the receptor.     

The effect of protein kinase C activation on G(z)-mediated regulation of type 2 and 6 adenylyl cyclases

Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2.     

Characterization of the G alpha(s) regulator cysteine string protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP(1-198) (full-length), CSP(1-112), and CSP(1-82) on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G alpha(s) and increases steady-state GTP hydrolysis. CSP(1-198) modulation of G alpha(s) was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP(1-112) was Hsc70-SGT-independent. CSP(1-112) preferentially associated with the inactive GDP-bound conformation of G alpha(s). Consistent with the stimulation of GTP hydrolysis, CSP(1-112) increased guanine nucleotide exchange of G alpha(s). The interaction of native G alpha(s) and CSP was confirmed by coimmunoprecipitation and showed that G alpha(s) associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the beta2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G alpha(s) and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.     

MANT-substituted guanine nucleotides: a novel class of potent adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G(s). 2'(3')-O-(N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G(i)/G(o)-proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTPgammaS) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on Galpha(s)- and Galpha(i)-mediated signaling. MANT-GTPgammaS and MANT-GppNHp had lower affinities for Galpha(s) and Galpha(i) than GTPgammaS and GppNHp. In contrast to guanosine 5'-[beta-thio]diphosphate, MANT-GTPgammaS noncompetitively inhibited GTPgammaS-stimulated AC in Galpha(s)-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPgammaS and MANT-GppNHp was not due to Galpha(s) inhibition since it was also observed in Galpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked Galpha(i)-mediated AC inhibition by GTPgammaS and GppNHp in S49 cyc(-) membranes but not AC inhibition by MANT-GTPgammaS and MANT-GppNHp. MANT-GTPgammaS and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.     

Interaction of small G proteins with photoexcited rhodopsin

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.     

Na+ promotes the dissociation between Galpha GDP and Gbeta gamma,activating G protein-gated K+ channels

G protein-gated K(+) channels (GIRK, or Kir3) are activated by the direct binding of Gbetagamma or of cytosolic Na(+). Na(+) activation is fast, Gbetagamma-independent, and probably via a direct, low affinity (EC(50), 30-40 mm) binding of Na(+) to the channel. Here we demonstrate that an increase in intracellular Na(+) concentration, [Na(+)](in), within the physiological range (5-20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na(+) effect. It is inhibited by a Gbetagamma scavenger, hence it is Gbetagamma-dependent; but it does not require GTP. We hypothesized that Na(+) elevates the cellular concentration of free Gbetagamma by promoting the dissociation of the Galphabetagamma heterotrimer into free Galpha(GDP) and Gbetagamma. Direct biochemical measurements showed that Na(+) causes a moderate decrease (approximately 2-fold) in the affinity of interaction between Galpha(GDP) and Gbetagamma. Furthermore, in accord with the predictions of our model, slow Na(+) activation was enhanced by mild coexpression of Galpha(i3). Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbetagamma-dependent regulation of GIRK by Na(+). We propose that Na(+) may act as a regulatory factor, or even a second messenger, that regulates effectors via Gbetagamma.     

The G protein alpha subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K(+) channels

In neuronal and atrial tissue, G protein-gated inwardly rectifying K(+) channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.     

Differential sensitivity of P-Rex1 to isoforms of G protein betagamma dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.