RESUMO
G gamma(13) is a divergent member of the G gamma 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 gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dimerização , Ativação Enzimática , Proteínas de Fluorescência Verde , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/classificação , Humanos , Indicadores e Reagentes/metabolismo , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Fosfolipase C beta , Isoformas de Proteínas , Subunidades Proteicas , Alinhamento de Sequência , Distribuição Tecidual , Fosfolipases Tipo C/metabolismoRESUMO
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.
Assuntos
Ciclo Celular , Hepatócitos/citologia , Fígado/citologia , Animais , Sequência de Bases , Linhagem Celular Transformada , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Primers do DNA , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Polimorfismo Conformacional de Fita Simples , Ratos , Ratos Endogâmicos F344 , Fuso Acromático , Proteína Supressora de Tumor p53/genéticaRESUMO
The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha 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 alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Compostos de Alumínio/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Técnicas Biossensoriais , Clonagem Molecular , Escherichia coli , Fluoretos/farmacologia , Guanosina Difosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos , Fases de Leitura Aberta , Proteínas RGS/química , Proteínas RGS/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas RGS/metabolismo , Proteínas RGS/fisiologia , Proteínas Repressoras , Transdução de Sinais , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Proteína Axina , Proteínas de Transporte/fisiologia , Ciclo Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/fisiologia , Proteínas/fisiologia , Homologia de Sequência de Aminoácidos , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
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.