Guanine Nucleotide Regulatory Protein Gs Research Articles

Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

BackgroundMalaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum. Methods and FindingsWe investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.ConclusionsTogether these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.

Muscarinic receptor regulation of cardiac adenylate cyclase activity

Stimulation and inhibition of adenylate cyclase activity are mediated by the guanine nucleotide regulatory proteins Gs and Gi, respectively. Two general mechanisms have been proposed for the inhibition of activated adenylate cyclase: direct inhibition of the catalyst by Gi, and indirect inhibition of the activated catalyst mediated by Gi inhibition of Gs. We have assessed direct inhibition of adenylate cyclase by evaluating the ability of Gpp(NH)p to inhibit the forskolin-stimulated enzyme in the presence of various concentrations of magnesium ions and the guanine nucleotide. Gpp(NH)p inhibition of adenylate cyclase activity was only observed in the presence of forskolin and low concentrations of MgCl2. Muscarinic agonists did not increase Gpp(NH)p inhibition of the forskolin-stimulated enzyme, even in the presence of low concentrations of MgCl2 and guanine nucleotide (near the respective Kact or Ki). Whether in the absence or presence of muscarinic agonists, no concentration of Gpp(NH)p was found to inhibit basal adenylate cyclase activity in the absence of forskolin. In addition, muscarinic agonists had no effect on the rate constant (kon) for Gpp(NH)p activation of the enzyme. In contrast to these data, the muscarinic agonist methacholine stimulated the inactivation rate constant (koff) for isoproterenol plus GTP-activated adenylate cyclase activity 15-fold, and the increase in koff was blocked by atropine. Moreover, the sarcolemma displayed specific, high affinity GTP hydrolytic activity which was stimulated by methacholine activation of muscarinic receptors. These data further support our original hypothesis, indicating that although direct inhibition of the catalyst by Gi may occur in cardiac sarcolemma, physiologically relevant attenuation of adenylate cyclase activity by muscarinic agonists occurs by a mechanism linked to GTP hydrolysis.