GTP-binding proteins Gi and Go transplanted onto Xenopus oocyte by rat brain messenger RNA

Abstract After injection with messenger RNA (mRNA) isolated from rat brain, Xenopus laevis oocytes acquired electrophysiological responsiveness to externally perfused acetylcholine (ACh) or serotonin (5-HT), and elevated responsiveness to internally applied guanosine 5′-(3- O -thio)triphosphate (GTPγS). Compared with the membranes of native oocytes, those of mRNA-injected oocytes contained increased amounts of 39 and 41 kDa proteins, which could be [ 32 P]ADP-ribosylated by pertussis toxin (PTX). The amplitude of the GTPγS response and the amounts of the 39 and 41 kDa proteins increased in a parallel manner for at least 3 days following mRNA injection. Current responses to internally applied GTPγS showed properties common to those of responses to ACh or 5-HT perfusion: both responses had reversal potentials close to the Cl − potential, were mimicked by intracellular injection of IP 3 , desensitized by a large dose of IP 3 , and inhibited by a simultaneous injection of neomycin or EGTA. Incubation of mRNA-injected cells with PTX inhibited both the 5-HT response and the [ 32 P]ADP-ribosylation of the 39 and 41 kDa proteins in a parallel, dose-dependent manner. After pretreatment of oocytes with PTX followed by mRNA injection, the levels of the 39 and kDa proteins and the 5-HT response appeared to be similar to those of non-treated cells injected with mRNA, whereas no detectable amounts of these proteins were induced when PTX-pretreated cells were analyzed under the same conditions without mRNA injection. From these results, we concluded that (1) PTX substrate 39 and 41 kDa G proteins were expressed in Xenopus oocytes by translation of injected rat brain mRNA, although native oocytes have their own G proteins, and (ii) the newly synthesized G proteins could also participate in the transplanted Ca 2+ -mobilizing receptor-mediated responses.