EVIDENCE FOR GUANOSINE TRIPHOSPHATE-BINDING PROTEINS IN TRYPANOSOMA-CRUZI

The transformation of the parasite Trypanosoma cruzi from the blood-bo rne trypomastigote to the intracellular amastigote constitutes a key c linical feature in the pathophysiology of Chagas' disease. That this t ransition occurs without change in the integrity of the plasma membran e of the parasite suggests the presence of biochemical structures, i.e ., signal transduction systems, that convey information regarding the external milieu of the host so as to facilitate this transformation. I n higher eukaryotes, it has been found that a heterotrimeric GTP-bindi ng protein (G-protein), composed of alpha beta gamma subunits, constit utes a critical component of this complex. Two closely related groups of G-proteins are substrates for cholera toxin (CT)- (Gs) and pertussi s toxin (PT)- (Gil-3 and Go) dependent ADP ribosylation. In concert, t hey link plasma membrane receptors to adenylate cyclase, resulting in the stimulation or inhibition, respectively, of cAMP generation. In th is report, we demonstrate the presence of bath groups of G-proteins. C holera toxin-dependent ADP ribosylation of 42- and 45-kD proteins was demonstrable in amastigotes (AMAST), in the cytosol of epimastigotes ( EPI), and weakly in trypomastigotes (TRYP), suggesting the presence of the stimulatory GTP-binding protein, Gs, in T. cruzi. Antisera genera ted against the as subunit of the Gs heterotrimeric protein (anti-alph a s) bound to a 45-kD protein CT substrate in the rank order TRYP >> A MAST approximate to EPI cytosol. Immunoprecipitation of CT-P-32-ADP-ri bosylated membranes with anti-alpha s resulted in 42- and 45-kD protei ns. However, no Gs-mediated activation of adenylate cyclase was demons trable in reconstitution studies using cyc(-) lymphoma cells, which la ck a functional Gs but possess a beta-adrenergic receptor and adenylyl cyclase enzyme. Pertussis toxin -catalyzed ADP ribosylation was demon strable in 39-40-kD particulate proteins of EPI, less strongly in AMAS T, and least in TRYP, consistent with the presence of inhibitory (Gi) and Go GTP-binding proteins. In support of this observation, immunoche mical analysis of the PT substrates identified the presence of alpha o and alpha(11-2-3) in EPI, AMAST and TRYP, although, with the exceptio n of alpha(13), both toxin and associated immunochemical PT substrates are decreased in AMAST and TRYP relative to EPI. Although the functio ns of these putative G-proteins in T. cruzi are still unclear, their e xpression may be regulated by the state of parasite differentiation. D espite our inability to demonstrate a function for these G-proteins in the adenylate cyclase signal transduction system, their presence in T . cruzi suggests that they may function in other signal transduction p athways yet to be elucidated. Future investigations of their function may reveal important targets for therapeutic intervention in this dise ase.