The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 7 nicotinic acetylcholine receptors mediate beta-amyloid peptide-induced tau protein phosphorylation

The Alzheimer's disease pathogenic peptide, beta-amyloid42 (A beta 42), induces tau protein phosphorylation. Because hyperphosphorylated tau is a consistent component of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease, we investigated the signaling molecules involved in A beta 42-induced tau phosphorylation. We show that A beta 42 elicited rapid and reversible tau protein phosphorylation on three proline-directed sites (Ser-202, Thr-181, and Thr-231) in systems enriched in alpha 7 nicotinic acetylcholine receptors (alpha 7nAChR) including serum-deprived human SK-N-MC neuroblastoma cells and hippocampal synaptosomes. Although alpha 7nAChR agonists induced similar phosphorylation, pretreatment with antisense-alpha 7nAChR oligonucleotides (in cells) or alpha 7nAChR antagonists (in cells and synaptosomes) attenuated A beta-induced tau phosphorylation. Western analyses showed that the mitogen-activated kinase cascade proteins, ERKs and c-Jun N-terminal kinase (JNK-1), were concomitantly activated by A beta 42, and their respective kinase inhibitors suppressed A beta-induced tau phosphorylation. More importantly, recombinant-activated ERKs and JNK-1 could differentially phosphorylate tau protein in vitro. Thus, the alpha 7nAChR may mediate A beta-induced tau protein phosphorylation via ERKs and JNK-1.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

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.     

RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.     

Selective role of G protein gamma subunits in receptor interaction

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy. However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5. Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai. These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.     

AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin

A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha. The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs. The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha. The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha. The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.     

Biochemical analysis of yeast G(alpha) mutants that enhance adaptation to pheromone

The mating-specific heterotrimeric G(alpha) protein of Saccharomyces cerevisiae, Gpa1, negatively regulates activation of the pheromone response pathway both by sequestering G(beta)gamma and by triggering an adaptive response through an as yet unknown mechanism. Previous genetic studies identified mutant alleles of GPA1 that downregulate the pheromone response independently of the pheromone receptor (GPA1E364K), or through a receptor-dependent mechanism (GPA1N388D). To further our understanding of the mechanism of action of these mutant alleles, their corresponding proteins were purified and subjected to biochemical analysis. The receptor-dependent activity of Gpa1N388D was further analyzed using yeast strains expressing constitutively active receptor (Ste2) mutants, and C-terminal truncation mutant forms of Gpa1. A combination of G(alpha) affinity chromatography, GTP binding/hydrolysis studies, and genetic analysis allowed us to assign a distinct mechanism of action to each of these mutant proteins.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Distinct interactions of GTP,UTP, and CTP with G(s) proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.     

The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.     

Hydrolysis of GTP by the alpha-chain of Gs and other GTP binding proteins

The functions of G proteins--like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)--depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein alpha-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the alpha-chain of Gs (alpha s) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of alpha s in human pituitary tumors inhibit the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in alpha s that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of GTPase inhibiting mutations in alpha s occurs in the codon for an Arg residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by alpha s. This Arg residue is located in a domain of alpha s not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.     

Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

beta-Amyloid-specific upregulation of stearoyl coenzyme A desaturase-1 in macrophages

beta-Amyloid peptide (A beta), a major component of senile plaques, the formation of which is characteristic of Alzheimer's disease (AD), is believed to induce inflammation of the brain mediated by microglia, leading to neuronal cell loss. In this study, we performed an oligonucleotide microarray analysis to investigate the molecular events underlying the A beta-induced activation of macrophages and its specific suppression by the A beta-specific-macrophage-activation inhibitor, RS-1178. Of the approximately 36,000 genes and expressed sequence tags analyzed, eight genes were specifically and significantly upregulated by a treatment with interferon gamma (IFN gamma) and A beta compared to a treatment with IFN gamma alone (p<0.002). We found that the gene for a well-characterized lipogenetic enzyme, stearoyl coenzyme A desaturase-1 (SCD-1), was specifically upregulated by A beta treatment and was suppressed to basal levels by RS-1178. Although the underlying mechanisms remain unknown, our results suggest the presence of a link between AD and SCD-1.     

Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity

The phospholipase C (PLC) pathway is the major signaling mechanism of photoactivation in invertebrate photoreceptors. Here we report the cloning of a cDNA encoding a 140-kDa retinal PLC that is uniquely expressed in squid photoreceptors. This cDNA encodes a protein with multiple distinct modular domains: PH, X and Y catalytic, and C2 domains, as well as G- and P-box motifs and two GTP/ATP binding motifs. The PLC was stimulated by activated squid Gq alpha but not by squid Gq beta gamma or mammalian beta gamma subunits. The PLC was inhibited by monophosphate, diphosphate and triphosphate nucleotides but not cyclic nucleosides. We also tested the ability of PLC-140 to regulate the GTPase activity of Gq alpha in the rhabdomeric membranes. Depletion of PLC-140 from the rhabdomeric membranes decreased the GTP hydrolysis but not GTP gamma S binding to the membranes. Reconstitution of purified PLC-140 with membranes accelerated Gq alpha GTPase activity by fivefold at a concentration of 2.5 microM. Our data suggest that PLC-140 plays an important role in both the activation and inactivation pathways of invertebrate visual transduction.     

Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn2+), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously [Verkman, A. S., Skorecki, K. L., & Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123] and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.     

Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.