Chan_2011_J.Biol.Chem_286_19932

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Galpha(q) and Galpha(s) regulation of neurotransmission, Galpha(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Galpha(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Galpha(i)/alpha(q)/alpha(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Galpha(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Galpha subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Galpha(s) and lesser amounts of Galpha(q) and Galpha(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to these subunits and Galpha(olf), whereas the Ric-8BDelta9 isoform stimulated Galpha(s short) GTPgammaS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Galpha(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Galpha(s) from approximately 385 nm to an estimated value of approximately 42 muM. Directionality of the Ric-8BFL-catalyzed Galpha(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Galpha(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B.Galpha nucleotide-free intermediate to release free Ric-8B and Galpha-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Galpha-GTP in cells.