Mammalian Ric‐8A and Ric‐8B are Required for Functional Expression of Heterotrimeric G‐Proteins

Ric‐8A and Ric‐8B are non‐GPCR GEFs for G protein α subunits and positive regulators of G protein signaling. To understand the regulatory role of Ric‐8s on G protein function, we derived murine Ric‐8A or Ric‐8B knockout (KO) embryonic stem cell lines. Steady‐state expression of Gαi, Gαq, Gα13, and total Gβ was reduced >95% in the Ric‐8A, but not Ric‐8B KO. Gαs expression was reduced 50 and 70% in the Ric‐8A and B KOs respectively, but basal and isoproterenol‐stimulated adenylyl cyclase (AC) activity was more attenuated in the Ric‐8A KO. We propose that reduced Gβγ levels in the Ric‐8A KO were a secondary consequence of the overall reduced bulk pool of Gα and accounted for the more dramatic AC activity reduction. Gene rescue of the Ric‐8A KO restored proper G protein expression and signaling. Initial Gαi and Gβ protein synthesis and Gi trimer formation proceeded normally in the Ric‐8A KO, but nascent Gαi and Gβ had markedly faster turnover rates (t1/2 ~2–3h) when compared to the Ric‐8A‐rescued cell line (t1/2 ~15h). Plasma membrane localization of GFP‐ or YFP‐Gαi, Gαq, Gαs, and Gβ1 persisted in both Ric‐8 KOs and a WT cell line, but the levels of each G protein were reduced dramatically in the Ric‐8A KO. YFP‐Gαs was reduced specifically in the Ric‐8B KO. We conclude that Ric‐8 proteins functionally protect the Gα subunits that each bind from undergoing rapid degradation, but do not direct G protein plasma membrane forward trafficking.