Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin.

Abstract

We have examined the isolated postsynaptic density (PSD) fraction for the presence of a G protein. First, we found specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate to the PSD. Second, pertussis toxin-activated ADP-ribosylation of the isolated PSD fraction resulted in the appearance of a G protein with an apparent molecular mass of 41 kDa, and two G proteins with apparent molecular masses of 41 kDa and 39 kDa in synaptic membrane (SM) fraction and total homogenate (H). The amount of the 41-kDa G protein per unit protein was in the order of SM greater than H greater than PSD. Anti-G(i0 antibodies recognized the 41-kDa G protein in both PSD and SM, whereas anti-G(o) antibodies reacted with the 39-kDa G protein in the SM. The absence of G(o) protein in the PSD suggested that there was no contamination with SM. Moreover, unlabeled PSD incubated with an extract of SM that contained the labeled G proteins resulted in no label in the subsequently reisolated PSD, suggesting that the G protein found in the PSD was not due to adsorption of the G protein onto the PSD during its isolation from the SM. PSD pretreated with EGTA gave an 11-fold increase in the ADP-ribosylation reaction of the G(i) protein; similar effects on the G(i) and G(o) proteins of SM were obtained. Restoration of Ca2+/calmodulin to the PSD, but not of either Ca2+ or calmodulin alone, removed the effect of EGTA, indicating a strong complex formation between G(i) and Ca2+/calmodulin that decreased the ADP-ribosylation reaction. Preincubation with the Ca(2+)-channel blocker nifedipine decreased the ADP-ribosylation reaction in the PSD. We conclude that G(i) is present in the PSD, that it may interact with calmodulin and that it is involved in the regulation of voltage-dependent Ca2+ channel. We present a theory of the involvement of the G protein and calmodulin in postsynaptic neurophysiological events.