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

Abstract

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including G␣ q and G␣ s regulation of neurotransmission, G␣ idependent mitotic spindle positioning during (asymmetric) cell division, and G␣ 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␣ i /␣ q /␣ 12/13 subunits. Ric-8B potentiated G s signaling presumably as a G␣ 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␣ subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed G␣ s and lesser amounts of G␣ q and G␣ 13 . Ric-8BFL stimulated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding to these subunits and G␣ olf , whereas the Ric-8B⌬9 isoform stimulated G␣ s short GTP␥S binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V max of G␣ s steady state GTP hydrolysis and the apparent K m values of GTP binding to G␣ s from ϳ385 nM to an estimated value of ϳ42 M. Directionality of the Ric-8BFL-catalyzed G␣ 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␣ s tightly, and near-K m GTP levels were required to dissociate the Ric-8B⅐G␣ nucleotide-free intermediate to release free Ric-8B and G␣-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce G␣-GTP in cells.Heterotrimeric G proteins transduce signals received from ligand-bound G protein-coupled receptors (GPCRs) 2 to intracellular effector enzymes. Agonist-bound GPCRs activate coupled G protein heterotrimers by accelerating the rate of GDP for GTP exchange on the G␣ subunit (1). The understanding of G protein signaling pathway complexity expanded beyond this traditional paradigm when modulatory proteins that regulate G protein activation apart from receptors were uncovered and characterized (2-7).One well characterized non-receptor G protein activator is Ric-8 (resistance to inhibitors of cholinesterase 8A). ric-8 was first identified in a genetic screen devised to find mutants of genes that positively regulated Caenorhabditis elegans neurotransmission (8). Through genetic epistasis analyses, it was predicted that ric-8 action was elicited upstream of G␣ q and/or G␣ s to regulate divergent G protein signaling outputs (8 -10). Ric-8 was first linked physically to G proteins when two homologous mammalian Ric-8 proteins (so-named Ric-8A and Ric-8B) were isolated in yeast two hybrid screens using G␣ o and G␣ s baits, respectively. Ric-8A interacted with G␣ i/o , G␣ q , and G␣ 13 subunits (7). Ric-8B interacted with G␣ s and G␣ q (7, 11). Evidence of a preferred interaction of Ric-8A with G␣ i -GDP led to experimentation showing that Ric-8A was a guanine nucleotide exchange factor for the monomeric G␣...

Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits

“…These Ric-8/ Gα combinations were chosen because of the known specificity of Ric-8-regulated Gα biosynthesis in cells and Gα subunit guanine nucleotide exchange stimulatory activities (14,19,20). The rate of translation and final abundances of Gαq and Gαolf were found to be nearly identical among the Ric-8A-, Ric-8B-, and mockdepleted RRL preparations (Fig.…”

“…Ric-8 proteins have greatly diminished affinity for Gα-GTP(γS) or Gα-GDP-AlF 4 − (19,20). To test this directly, purified Gαq-GTPγS (100 nM) was prepared as described and incubated with or without 1 μM Ric-8A (33).…”

“…Ric-8 was discovered in Caenorhabditis elegans and implicated to genetically interact with various Gα subunits (15)(16)(17)(18). Mammalian Ric-8 proteins were then defined as Gα subunit guanine nucleotide exchange factors (GEFs) (19,20). Ric-8A and Ric-8B collectively stimulate nucleotide exchange of all Gα subunit classes by stabilizing the Gα nucleotide-free transition state.…”

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

“…In cells, agoniststimulated 7-transmembrane helical G protein-coupled receptors (GPCRs) catalyze nucleotide exchange from G protein heterotrimers, in which Gα•GDP is bound to a heterodimer of Gβ and Gγ subunits (5). The cytosolic proteins Ric-8A and Ric-8B, which are structurally unrelated to GPCRs, have been shown to have guanine nucleotide exchange (GEF) activity toward Gα•GDP subunits in the absence of Gβγ (6,7), thus functionally activating the subunit. In Caenorhabditis elegans, Drosophila, and mouse, Ric-8 homologs have been shown to be essential for asymmetric cell division, where they are assumed to function as GEFs (8)(9)(10)(11)(12).…”

ARMS/Kidins220 and synembryn-B levels regulate NGF-mediated secretion

“…Ric-8 works as a non-canonical guanidine exchange factor (GEF) for monomeric Gα proteins (Klattenhoff et al, 2003;Tall et al, 2003). In mammals, the orthologs of Ric-8, named synembryn-A and synembryn-B, exhibit different specificities for Gα proteins (Chan et al, 2011;Romo et al, 2008). In addition to modulating phospholipase C (PLC)β through Gαq, the C. elegans Ric-8 activates a parallel pathway through activation of Trio, a GEF for Rho GTPases (Williams et al, 2007).…”