Doctor of Philosophy, University of Rochester (2012)
Master of Science, University of Rochester (2009)
Bachelor of Science, Ort Horowitz (2007)
Group II activators of G-protein signaling (AGS) serve as binding partners for G?(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-G? signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with G?(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G?(i1)-YFP BRET were regulated by Ric-8A but not by G?-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and G?(i1)-YFP. The inhibitory regulation of GPR-G?(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-G?(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-G?(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G-G?(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-G?(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore G? expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, G?(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.
View details for DOI 10.1074/jbc.M112.418467
View details for Web of Science ID 000314397900011
View details for PubMedID 23212907
Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide-binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A- or Ric-8B-deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A(-/-) ES cells, which resulted from reduced steady-state amounts of G?(i), G?(q), and G?(13) proteins to <5% of those of wild-type cells. The amounts of G?(s) and total G? protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of G?(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein ? subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous G?(i) and G?(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein ? subunits that reached the membrane still bound to nascent G??. Finally, G?(i), G?(q), and G?(1) proteins exhibited accelerated rates of degradation in Ric-8A(-/-) cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent G? subunits with cellular membranes.
View details for DOI 10.1126/scisignal.2002223
View details for Web of Science ID 000297372000007
View details for PubMedID 22114146
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?(i)-dependent 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 ?385 nm to an estimated value of ?42 ?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-8B·G? 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.
View details for DOI 10.1074/jbc.M110.163675
View details for Web of Science ID 000291027700072
View details for PubMedID 21467038
Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein ? subunits. Co-expression of G? subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G? protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein ? subunit purification that was applicable to all G? subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G?(olf) for the first time and unprecedented yield of G?(q) and G?(13). G? subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·G? complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G? subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[?-thio]triphosphate (GTP?S), and stimulate appropriate G protein effector enzymes. A primary characterization of G?(olf) showed that it binds GTP?S at a rate marginally slower than G?(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G?(s short).
View details for DOI 10.1074/jbc.M110.178897
View details for Web of Science ID 000286464300028
View details for PubMedID 21115479