title: Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor.
abstract: ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(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 Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ∼385 nm to an estimated value of ∼42 μM. Directionality of the Ric-8BFL-catalyzed Gα(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 Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: PuiYee
authors.initials: P
authors.lastname: Chan
authors.forename: Meital
authors.initials: M
authors.lastname: Gabay
authors.forename: Forrest A
authors.initials: FA
authors.lastname: Wright
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Tall
mesh_terms: D000818:Animals; D002384:Catalysis; D002460:Cell Line; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms; D051381:Rats
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D020033:Protein Isoforms; C485884:Ric8a protein, mouse; C559967:Ric8b protein, mouse; D006160:Guanosine Triphosphate