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PubMed1990

Lithium-selective alteration of the function of brain versus cardiac Gs protein.

Lithium was recently demonstrated to inhibit the coupling of both muscarinic cholinergic receptors and beta-adrenergic receptors to pertussis toxin-sensitive and cholera toxin-sensitive G proteins, respectively, thus suggesting alteration of the function of G protein by lithium, as the single site for both the antimanic and antidepressant effects of this drug. One of the most puzzling aspects of the ability of lithium to ameliorate the manic-depressive condition, is its relatively selective action upon the central nervous system (CNS). In the present study, it was shown that lithium selectively attenuated the function of Gs proteins in the CNS, assessed through isoproterenol-induced increases in the binding of guanosine triphosphate (GTP) to these proteins. Therapeutic concentrations of lithium (1-1.5 mM in vitro) inhibited the function of Gs protein in the cerebral cortex of the rat, while 4- to 6-fold larger concentrations of lithium were required to alter the function of Gs protein equivalently in the cardiac ventricles of the rat. Chronic administration of lithium via rat chow, containing lithium carbonate, to rats totally abolished the effect of isoproterenol on the binding of GTP in the CNS but did not affect the function of peripheral cardiac Gs protein. The lithium-selective action on the function of Gs protein in the CNS may stem from the heterogeneity of the alpha s subunit proteins: in the heart, the major species is a 45 kDa molecule, while in the brain, a 52 kDa molecular weight species predominates. The heterogeneity in alpha s subunits may thus be the biochemical basis for the selective action of lithium on the CNS and for the scarcity of peripheral side effects.

Adenosine Diphosphate Ribose↗

PubMed1995

The alpha-subunit mRNAs for Gs and Go2 are differentially regulated by protein kinase A and protein kinase C in rat Sertoli cells.

In the present study, we have examined regulatory effects of protein kinase A and protein kinase C activation by 8-CPTcAMP and TPA, respectively, on mRNAs for various G protein alpha-subunits and corresponding immunoreactive proteins in rat Sertoli cells. Gs alpha and Go alpha mRNA levels were transiently increased 1.5-fold and 4-fold, respectively, by 8-CPTcAMP in cultured Sertoli cells. This up-regulation of mRNAs for Gs alpha and Go alpha was also observed when Sertoli cells were incubated in the presence of FSH. When protein synthesis was inhibited by cycloheximide, the cAMP-mediated stimulation of Gs alpha mRNA was abolished, whereas Go alpha mRNA was superinduced to a 50- to 100-fold higher level than basal. Activation of protein kinase C with TPA had a strong, synergistic effect on cAMP-mediated stimulation of Gs alpha mRNA, whereas the cAMP-mediated stimulation of Go alpha mRNA was completely blocked. Surprisingly, changes in mRNA levels were not accompanied by any alterations in the levels of immunoreactive Gs alpha and Go alpha proteins.

Animals↗

PubMed1994

The effect of chronic morphine and cocaine administration on the Gs and Go protein messenger RNA levels in the rat hippocampus.

The effects of single (20 or 50 mg/kg) and repeated morphine (14 days, rising doses: four days, 2 x 10 mg/kg; two days, 2 x 20 mg/kg; two days, 2 x 30 mg/kg; two days, 2 x 40 mg/kg and four days, 2 x 50 mg/kg i.p.) or cocaine (20 mg/kg i.p., once every hour for 3 h for one or five days) on the Gs alpha and Go alpha protein messenger RNA levels were investigated in the rat hippocampus. As shown by in situ hybridization immunocytochemistry and optical densitometry of autoradiograms, acute morphine (20 mg/kg) decreased the Gs alpha messenger RNA level and increased the Go alpha messenger RNA level in the dentate gyrus and CA1 hippocampal region. Repeated morphine administration significantly decreased the hippocampal Gs alpha messenger RNA level at 12, 24 and 48 h, and enhanced the Go alpha messenger RNA level in those areas at 12 and 24 h after the last injection. Repeated and acute cocaine administration decreased the Go alpha messenger RNA level in the CA3 region and increased it in the CA1 at various time points. Furthermore, in all hippocampal regions, acute, but not chronic, cocaine administration increased the Gs alpha mRNA level at 24 h, whereas chronic treatment significantly decreased that level at 48 h after the last dose. The data indicate that chronic morphine elevates the Go and attenuates the Gs protein biosynthesis at later time points in all regions of the hippocampal formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS).

Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells. Higher concentrations of cholera-toxin reversed this effect. Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme. Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells. Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation. The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel. Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment. These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.

Adenosine Diphosphate Ribose↗

PubMed1990

Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs).

Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [32P]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of Gs alpha. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD+ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of Gs alpha demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for Gs alpha as neither the alpha-subunits of the pertussis toxin-sensitive G-proteins Gi2 and Gi3, nor the beta subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of Gs alpha nor of the alpha-subunits of Gi2 or Gi3 although it did cause ADP ribosylation of the entire complement of both Gi2 and Gi3 in the membranes. Despite the reduction in levels of immunoreactive Gs alpha from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted.

Adenosine Diphosphate Ribose↗

PubMed1996

Gs protein mutations and the pathogenesis and function of pituitary tumors.

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) production and somatotroph proliferation by binding to a seven transmembrane-domain receptor, linked to Gs. Gs stimulates production of cyclic adenosine 3'-5'-monophosphate (cAMP) and hence activation of protein kinase A (PKA). A subgroup of pituitary somatotroph adenomas has been demonstrated, which has constitutive activation of Gs, with reduced in vitro responsiveness to agents that stimulate Gs. Subsequently, somatotroph adenomas have been identified, which have activating mutations of Gs (gsp). However, there are no clear clinical or biochemical phenotypic characteristics that enable gsp-positive and gsp-negative tumors to be differentiated from one another. Gsp mutations occur in 35% to 40% of somatotroph adenomas in caucasians, but have a much lower reported prevalence of 4% to 9% in the Japanese population. G-protein mutations also occur in clinically nonfunctioning pituitary tumors and, rarely, in corticotroph adenomas. There is little direct evidence at present to suggest that the gsp mutation has a primary oncogenic role in the pathogenesis and function of pituitary tumors. Further functional studies are needed. The gsp mutation is probably one of several oncogenic mutations required for pituitary tumor development.

GTP-Binding Proteins↗

PubMed1986

Effect of Al3+ plus F- on the catecholamine-stimulated GTPase activity of purified and reconstituted Gs.

The effects of Al3+ and F- on the catecholamine-stimulated GTPase cycle were studied by using reconstituted phospholipid vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. Al3+/F- activated reconstituted Gs to levels previously reported for detergent-solubilized, purified Gs, although both activation and deactivation were faster in the reconstituted preparation. Under these conditions, Al3+/F- did not inhibit by more than 15% the beta-adrenergic agonist-stimulated GTPase activity of the vesicles nor did it significantly inhibit the rates of GTP binding, GTP hydrolysis, or GDP release. When Mg2+ (50 mM) was used instead of agonist to promote GTP hydrolysis in the receptor-Gs vesicles, Al3+/F- was found to inhibit GTP gamma S binding, GDP release, and steady-state GTPase activity to unstimulated levels. These data can be interpreted as indicating that the receptor catalyzes nucleotide exchange by Gs faster or more efficiently than does Mg2+.

Aluminum↗

PubMed1990

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha.

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed2004

Role of amino acid residues on the GS region of Stichopus arginine kinase and Danio creatine kinase.

Stichopus arginine kinase (AK) is a unique enzyme in that it evolved not from the AK gene but from the creatine kinase (CK) gene: the entire amino acid sequence is homologous with other CKs apart from the guanidine specificity region (GS region), which is identical in structure to that of AK. Ten independent mutations were introduced around the GS region in Stichopus AK. When an insertion or deletion was introduced near the GS region, the Vmax of the mutant enzyme was dramatically decreased to less than 0.1% of the wild type, suggesting that the length of the GS region is crucial for the recognition of the guanidine substrate. Replacement of Phe63 and Leu65 to Gly in the Stichopus enzyme caused a remarkable increase in the Kmarg. This indicates that Phe63 and Leu65 are associated with the arginine substrate-binding affinity. The hydrogen bond formed between the Asp62 and Arg193 residues is thought to play a key role in stabilizing the closed substrate-bound structure of AK. Mutants that eliminated this hydrogen bond had a considerably decreased Vmax, accompanied by a threefold increase in Kmarg. It is noted that the value of the Kmarg of the mutants became very close to the Kdarg value of the wild type. Six independent mutations were introduced in the GS region of Danio M-CK. Almost equivalent values of Kmcr and Kdcr in all of the mutants indicated that a typical synergism was completely lost. The results suggested that the Ile69 to Gly mutant, displaying a high Kmcr and a low Vmax, plays an important role in creatine-binding. This is consistent with the observation that in the structure of Torpedo CK, Ile69 provides a hydrophobic pocket to optimize creatine-binding.

Amino Acid Sequence↗

PubMed2000

Effects of diadenosine polyphosphates (Ap(n)As) and adenosine polyphospho guanosines (Ap(n)Gs) on rat mesenteric artery P2X receptor ion channels.

Diadenosine polyphosphates (Ap(n)As, n=3 - 7) and adenosine polyphospho guanosines (Ap(n)Gs, n=3 - 6) are naturally occurring vasoconstrictor substances found in platelets. These vasoconstrictor actions are thought to be mediated through the activation of P2X receptors for ATP. The effects of Ap(n)As and Ap(n)Gs at P2X receptors on rat mesenteric arteries were determined in contraction studies and using the patch clamp technique on acutely dissociated artery smooth muscle cells. P2X(1) receptor immunoreactivity was detected in the smooth muscle layer of artery rings. The sensitivity to alpha,beta-methylene ATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A evoked concentration dependent P2X receptor inward currents which desensitized during the application of higher concentrations of agonist. The agonist order of potency was Ap(5)A> or = Ap(6)A> or = Ap(4)A >> Ap(3)A. Ap(2)A and Ap(7)A were ineffective. Similar results were obtained in contraction studies except for Ap(7)A which evoked a substantial contraction. Ap(n)Gs (n=2 - 6)(30 microM) evoked P2X receptor inward currents in mesenteric artery smooth muscle cells. Ap(n)Gs (n=4 - 6) were less effective than the corresponding Ap(n)A. This study shows that at physiologically relevant concentrations Ap(n)As and Ap(n)Gs can mediate contraction of rat mesenteric arteries through the activation of P2X(1)-like receptors. However the activity of the longer chain polyphosphates (n=6 - 7) may be overestimated in whole tissue studies due to metabolic breakdown to yield the P2X receptor agonists ATP and adenosine tetraphosphate. British Journal of Pharmacology (2000) 129, 124 - 130

Animals↗

PubMed1989

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

Animals↗

PubMed1989

Guanine-nucleotide-binding proteins Gi and Gs in fat-cells from normal, hypothyroid and obese human subjects.

In human adipocyte plasma membranes, pertussis toxin catalysed the ADP-ribosylation of an apparently single 40 kDa protein. The same protein was also observed in Western blots by using an antibody which identifies the C-terminal decapeptide of Gi alpha (alpha subunit of Gi). In analogous experiments, cholera toxin and an antibody raised against the C-terminal decapeptide of Gs alpha (alpha subunit of Gs) were used to identify two proteins of 42 and 45 kDa, the former of which was more prominent. A method was developed to estimate the relative amounts of Gi and Gs in crude adipocyte plasma membranes in a single immunoblot by using the two antisera. In animal models, changes in the amounts of G-proteins have been suggested to explain alterations in hormone-responsiveness in hypothyroidism and obesity. However, the amounts of Gi and Gs were unaltered in thyroidectomized papillary-carcinoma patients who had been without hormone substitution for 4 weeks. In adipocyte plasma membranes prepared from markedly obese subjects, the amounts of both Gi alpha and Gs alpha as calculated per mg of protein were decreased, but the Gi/Gs ratio remained unaltered in comparison with control subjects.

Adipose Tissue↗

PubMed1993

Correlation between prolactin secretion and Gs protein expression during sustained cholera-toxin stimulation.

We have studied the chronic effect of cholera toxin (CTX) on prolactin synthesis and secretion in GH3 pituitary-tumour cells. Time-course analysis showed that prolactin secretion increased with time of CTX exposure, reached a peak at 3 h, and decreased thereafter. Prolactin synthesis was also shown to be stimulated by CTX. The basic and forskolin-stimulated cyclic AMP levels of the CTX-treated cells followed a biphasic time response similar to that of prolactin secretion. Exposure of cells to CTX for more than 3 h abolished the subsequent CTX-catalysed ADP-ribosylation in vitro. Moreover, a significant decrease in the pertussis-toxin-catalysed ADP-ribosylation was found after cells were exposed to CTX for longer than 6 h. Western-blot analysis indicated that the amount of Gs alpha (alpha-subunit of Gs) protein increased within 3 h, followed by a gradual decrease to 50% of the control level at 24 h. The accumulation of Gs alpha mRNA increased within 6 h of CTX exposure, and decreased thereafter to 40% of the basal level at 48 h. Our findings that prolonged treatment of CTX induced similar patterns of time responses in Gs alpha protein expression, cyclic AMP production and prolactin secretion indicate that CTX-induced changes in Gs alpha protein levels may be responsible for the cellular response leading to prolactin secretion.

Adenosine Diphosphate Ribose↗

PubMed2002

Expression of excitatory amino acid transporter-2 (EAAT-2) and glutamine synthetase (GS) in brain macrophages and microglia of SIVmac251-infected macaques.

Na+-dependent transporters for glutamate (excitatory amino acid transporters, EAATs) clear extracellular glutamate in the brain and prevent excitotoxic neuronal damage. Glutamine synthetase (GS) provides metabolic support for neurones by producing the neurotrophic amino acid glutamine. EAAT and GS expression has recently been demonstrated in macrophages and microglial cells in vitro, and in two models of acute inflammation in vivo. This observation might modify our current understanding of brain inflammation, which considers activated microglia and brain macrophages as the main neurotoxic cells through their production of a variety of neurotoxins, including glutamate. EAAT and GS expression by these cells would entail neuroprotective and neurotrophic properties, counterbalancing the deleterious consequences of microglial activation. Macaque infection by the simian immunodeficiency virus (SIV) is considered the most relevant model for human acquired immunodeficiency syndrome (AIDS), including chronic inflammation of the brain at the early asymptomatic stage of the infection, followed by an AIDS-like disease where neuronal death occurs. We studied the expression of EAAT-2 and GS in the brains of three SIVmac251-infected and two noninfected cynomolgus macaques. We found that both microglia and brain macrophages expressed EAAT-2 and GS in infected primates, suggesting that these cells might, like astrocytes, clear extracellular glutamate and provide glutamine to neurones. Microglia and macrophages could thus have neuroprotective and neurotrophic properties in addition to their production of neurotoxins. This finding might explain the contrast between early intense microglial activation and the late occurrence of neuronal apoptotic cell death, which is mainly observed at the terminal stage of the disease.

Animals↗

PubMed1986

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.

Animals↗

PubMed1989

Corticosterone differentially regulates the expression of Gs alpha and Gi alpha messenger RNA and protein in rat cerebral cortex.

The possibility that glucocorticoids regulate specific guanine nucleotide binding regulatory proteins (G proteins) was investigated in rat cerebral cortex. Corticosterone was administered to normal and bilaterally adrenalectomized rats, and hormone regulation of individual G-protein subunits was investigated in cerebral cortex in three ways: (i) immunoblot analysis of subunit protein, (ii) hybridization blot analysis of subunit mRNA, and (iii) ADP-ribosylation analysis of stimulatory G protein (Gs alpha) subunits. Chronic (7 days) corticosterone administration to normal rats increased levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation but decreased levels of inhibitory G protein (Gi alpha) mRNA and tended to decrease levels of Gi alpha immunoreactivity. In contrast, levels of Go alpha and G beta immunoreactivity and mRNA were not influenced by corticosterone treatment. In adrenalectomized rats, corticosterone treatment produced a 25-50% increase in the levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation, whereas the hormone produced a 20-35% decrease in the levels of Gi alpha immunoreactivity and mRNA. Adrenalectomy, without corticosterone replacement, produced the opposite effects on Gs alpha and Gi alpha compared to sham-operated controls, indicating that these G proteins are regulated by this class of steroid hormone under physiological conditions in vivo. The results indicate that specific G-protein subunits--namely, Gs alpha and Gi alpha--are under the coordinated control of glucocorticoids in rat brain and demonstrate that G proteins are physiological targets of glucocorticoids in vivo. Possible roles played by these G-protein responses in mediating the effects of glucocorticoids on brain function are discussed.

Adenosine Diphosphate Ribose↗

PubMed1991

Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit.

Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.

Amino Acid Sequence↗

PubMed1996

Two cytoplasmic domains of mammalian adenylyl cyclase form a Gs alpha- and forskolin-activated enzyme in vitro.

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2). The first cytoplasmic domain of type I enzyme (IC1) and the second cytoplasmic domain of type II enzyme (IIC2-delta 3, a construct in which 36 N-terminal amino acids of the C2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in Gs alpha- and forskolin-activated enzyme activity. The turnover number for Gs alpha- and forskolin-stimulated enzyme activity of the complex between IC1 and IIC2-delta 3 was 8.2 s-1. The concentration of IIC2-delta 3 to achieve half-maximal activation of IC1 was 0.8 and 1.3 microM when stimulated by forskolin and Gs alpha, respectively. The concentration of IIC2-delta 3 needed to complex with IC1 was reduced 10-fold (0.08 microM) when the enzyme was activated by both forskolin and Gs alpha, suggesting that Gs alpha and forskolin increased the affinity of the two cytoplasmic domains for each other.

Adenylyl Cyclases↗