Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including G alpha(q) and G alpha(s) regulation of neurotransmission, G alpha(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and G alpha(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 G alpha(i)/alpha(q)/alpha(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a G alpha(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be G alpha subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed G alpha s and lesser amounts of G alpha(q) and G alpha(13). Ric-8BFL stimulated guanosine 5 '-3-O-(thio) triphosphate (GTP gamma S) binding to these subunits andG alpha(olf), whereas the Ric-8B Delta 9 isoform stimulated G alpha(s) (short) GTP gamma S binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V-max of G alpha(s) steady stateGTPhydrolysis and the apparent K-m values of GTP binding to G alpha(s) from similar to 385 nM to an estimated value of similar to 42 mu M. Directionality of the Ric-8BFL-catalyzed G alpha 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 G alpha s tightly, and near-K-m GTP levels were required to dissociate the Ric-8B.G alpha nucleotide-free intermediate to release free Ric-8B and G alpha-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce G alpha-GTP in cells.