Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3.
Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.
Thomas
Celestine J
CJ
Division of Biological Science, University of Montana, Missoula, Montana 59812, USA.
Tall
Gregory G
GG
Adhikari
Anirban
A
Sprang
Stephen R
SR
D000818:Animals; D001667:Binding, Competitive; D002151:Calorimetry / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry*; D002384:Catalysis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006147:Guanine / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D007700:Kinetics; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D016366:Open Reading Frames; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D002352:Carrier Proteins; C498913:Gpsm1 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C485885:Ric8a protein, rat; D006153:Guanosine Diphosphate; D006147:Guanine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go