| Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric G protein signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A-- or Ric-8B--deleted embryonic stem (ES) cell lines from blastocysts of these mice.;We observed pleiotropic G protein signaling defects in Ric-8A -/- ES cells, which resulted from reduced steady-state amounts of Galphai, Galphaq, and Galpha13 proteins to <5% of those of wild-type cells. Only the amount of Galphas was reduced substantially in Ric-8B-/- cells. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Galphai and Galphaq were efficiently translated in Ric-8A-/- cells but integrated into endomembranes poorly; however, the reduced amounts of Galpha subunits that reached the membrane still bound to nascent Gbetagamma. Interestingly, the Galphai and Galphaq proteins that remained soluble in the Ric-8A-/- cells, had a higher apparent molecular weight, suggestive towards the presence of a putative post translational modification (PTM) that is revealed when Ric-8A is absent. In addition, Galphai, Galphaq, and Gb1 proteins exhibited accelerated rates of degradation in Ric-8A-/- cells compared to those in wild-type cells.;Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Galpha subunits with cellular membranes. From further investigation of the slower migrating Galpha proteins we concluded that in the absence of Ric-8 proteins, newly synthesized Galpha proteins are not properly folded and become resistant to an artifactual proteolytic event that occurs by cellular proteases. In addition, we found that Ric-8A is natively phosphorylation in mammalian cells, however some site directed mutations of predicted phosphorylated sites resulted in full rescue of G protein expression while others generated a Ric-8A protein that was unstable when expressed in ES or insect cells. We believe that phosphorylation or dephosphorylation of Ric-8A has a functional purpose that can reveal further mechanistic information in regards to the folding and initial trafficking of Galpha proteins, as well as the discovery of cellular Ric-8 activators. |
