The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein ␣ subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G␣ i/o -Loco (GoLoco) motif predicted to represent a second G␣ interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G␣ i1 , G␣ i2 , and G␣ i3 in their GDPbound forms. In GTP␥S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G␣ i1 . Both regions also stabilize G␣ i1 in its GDPbound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF 4 Ϫ . Our results indicate that both RGS12 and RGS14 harbor two distinctly different G␣ interaction sites: a previously recognized N-terminal RGS box possessing G␣ i/o GAP activity and a C-terminal GoLoco region exhibiting G␣ i GDI activity. The presence of two, independent G␣ interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G␣ GAP activity.In the standard model of heterotrimeric G-protein signaling, cell surface receptors (GPCRs) 1 are coupled to a membraneassociated heterotrimer composed of G␣, G␤, and G␥ subunits (1, 2). G␤ and G␥ form an obligate heterodimer that binds tightly to GDP-bound G␣ subunits, enhancing G␣ coupling to receptor and inhibiting its release of GDP (i.e. G␤␥ dimers exhibit "guanine nucleotide dissociation inhibitor" (GDI) activity; Refs. 3-5). Upon agonist binding, the GPCR becomes a guanine nucleotide exchange factor (GEF) and promotes replacement of bound GDP for GTP on the G␣ subunit. The binding of GTP changes the conformation of three "switch" regions within G␣, allowing G␤␥ dissociation. GTP-bound G␣ and free G␤␥ subunits both initiate signals by interactions with downstream effector proteins until the intrinsic guanosine triphosphatase (GTPase) activity of G␣ returns the protein to the GDP-bound state. Reassociation of G␤␥ with GDP-bound G␣ obscures critical effector contact sites and terminates all effector interactions (6, 7). Hence, the duration of heterotrimeric G-protein signaling is controlled by the guanine nucleotide state of the G␣ subunit.We and others have identified a family of GTPase-activating proteins (GAPs) for G␣ subunits, the "regulators of G-protein signaling" or RGS proteins (8 -11). These proteins all contain a hallmark "RGS box," which accelerates the intrinsic GTPase rate of G␣ subunits by binding avidly to the transition state for GTP hydrolysis (12). Discovery of RGS box-mediated GAP activity finally resolved the paradox that GPCR-stimulated signals terminate much faster in vivo than predicted from the slow GTP hydrolysis rates exhibited by purified G␣ subunits in vitro (13). However, R...

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits - the "regulator of G protein signaling" or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.

G␥ 13 is a divergent member of the G␥ subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G␥ 13 contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G␥-like (GGL) domains which dimerize exclusively with G␤ 5 subunits. In this study, we investigated the functional range of G␥ 13 assembly with G␤ subunits using multiple assays of G␤ association and G␤␥ effector modulation. G␥ 13 was observed to associate with all five G␤ subunits (G␤ 1-5 ) upon co-translation in vitro, as well as function with all five G␤ subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (␣ 1B ) calcium channels. Multiple G␤/ G␥ 13 pairings were also functional in cellular assays of phospholipase C (PLC) ␤2 activation and inhibition of G␣ q -stimulated PLC␤1 activity. However, upon cellular co-expression of G␥ 13 with different G␤ subunits, only G␤ 1 /G␥ 13 , G␤ 3 /G␥ 13 , and G␤ 4 /G␥ 13 pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G␥ 13 forms functional G␤␥ dimers with a range of G␤ subunits. Coupled with our detection of G␥ 13 mRNA in mouse and human brain and retina, these results imply that this divergent G␥ subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.One major class of cellular signal transduction pathways is controlled by heterotrimeric guanine nucleotide-binding proteins ("G proteins"). The conventional model of heterotrimeric G-protein signaling involves serpentine cell-surface receptors (G protein-coupled receptors) coupled to a membrane-associated heterotrimer composed of a GTP-hydrolyzing G␣ subunit and a G␤␥ dimeric partner (1, 2). The WD-repeat ␤-propeller protein G␤ and the ␣-helical isoprenylated polypeptide G␥ form an obligate heterodimer that binds tightly to GDP-bound G␣, enhancing G␣ coupling to receptor and inhibiting its release of GDP. Guanine nucleotide exchange activity of agonist-occupied G protein-coupled receptors facilitates dissociation of G␣⅐GTP and G␤␥ subunits and allows both moieties to modulate a variety of downstream effectors; for the free G␤␥ dimer, these effectors include the second messenger generators adenylyl cyclase and phospholipase C-␤ (PLC␤) 1 as well as ion channels such as G protein-coupled inward-rectifying potassium (GIRK) channels and N-type calcium channels (3, 4).Considerable functional diversity is possible within the large combinatorial range of potential G␤␥ dimers (5, 6), given the existence of at least six G␤ subunits (G␤ 1-4 , the outlier G␤ 5 , and its retinal-specific isoform G␤ 5L ; Refs. 7 and 8) and 11 G␥ subunits (three farnesylated and eight geranylgeranylated species; Fig. 1). However, outside the unique nature of G␥ 1 (the farnesylated G␥ of the retinal phototransduction cascade; Refs. 9 -12), specific roles for particular G␥ subunit...

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.