RESUMEN
After activation of G protein-coupled receptors, G protein ßγ dimers may translocate from the plasma membrane to the Golgi apparatus (GA). We recently report that this translocation activates extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) via PI3Kγ; however, how Gßγ-PI3Kγ activates the ERK1/2 pathway is unclear. Here, we demonstrate that chemokine receptor CXCR4 activates ADP-ribosylation factor 1 (ARF1), a small GTPase important for vesicle-mediated membrane trafficking. This activation is blocked by CRISPR-Cas9-mediated knockout of the GA-translocating Gγ9 subunit. Inducible targeting of different Gßγ dimers to the GA can directly activate ARF1. CXCR4 activation and constitutive Gßγ recruitment to the GA also enhance ARF1 translocation to the GA. We further demonstrate that pharmacological inhibition and CRISPR-Cas9-mediated knockout of PI3Kγ markedly inhibit CXCR4-mediated and Gßγ translocation-mediated ARF1 activation. We also show that depletion of ARF1 by siRNA and CRISPR-Cas9 and inhibition of GA-localized ARF1 activation abolish ERK1/2 activation by CXCR4 and Gßγ translocation to the GA and suppress prostate cancer PC3 cell migration and invasion. Collectively, our data reveal a novel function for Gßγ translocation to the GA to activate ARF1 and identify GA-localized ARF1 as an effector acting downstream of Gßγ-PI3Kγ to spatiotemporally regulate G protein-coupled receptor signaling to mitogen-activated protein kinases.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor 1 de Ribosilacion-ADP/análisis , Subunidades beta de la Proteína de Unión al GTP/análisis , Células HEK293 , Humanos , Proteínas Quinasas Activadas por Mitógenos/análisis , Células PC-3 , Multimerización de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/análisis , Transducción de SeñalRESUMEN
Like those in mammals, heterotrimeric G protein complexes have been implicated in signal transduction pathways in plants; however, the subunits themselves have not been isolated. In this study, the rice heterotrimeric G protein subunits α (Gα) and ß (Gß) were purified by affinity chromatography using anti-Gα and -Gß antibodies and SDS-PAGE. Six and seven peptides, respectively, were identified by mass spectrometry and identified as the translation products of the Gα gene RGA1 and Gß gene RGB1. During purification, the subunits dissociated easily from the G protein complex.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/análisis , Subunidades alfa de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Oryza/química , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Anticuerpos/inmunología , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/inmunología , Espectrometría de Masas , Datos de Secuencia Molecular , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Resinas Sintéticas/químicaRESUMEN
Cells co-express multiple G protein ß and γ subunit isoforms, but the extent to which individual subunits associate to form particular ßγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific ßγ complexes may have unique functions despite the fact that most complexes exhibit similar properties when assayed in reconstituted systems. This chapter describes how multicolor bimolecular fluorescence complementation (BiFC) can be used in living cells to study the association preferences of ß and γ subunits. Multicolor BiFC determines the association preferences of these subunits by quantifying the two fluorescent complexes formed when ß or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete for interaction with limiting amounts of a common γ or ß subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). One means by which ßγ complexes may differ from each other and thereby mediate unique functions in vivo is in the kinetics and patterns of their internalization responses to stimulation of G protein-coupled receptors (GPCRs). Methods are described for imaging and quantifying the internalization of pairs of ßγ complexes in response to GPCR stimulation in living cells.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas Luminiscentes/análisis , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/análisis , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Estructuras Celulares/metabolismo , Estructuras Celulares/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Characterization of G protein ßγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native ßγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and ßγ labeled with heavy isotopes purified. Heavy ßγ was combined with either recombinant ßγ purified from Sf9 cells, ßγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched ßγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing ß and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three ß isoforms, ß(1), ß(2), and ß(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. ß(1) and γ(5) were most abundant in the enriched ßγ fraction, and this ßγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more ß(4) and γ(5) compared to the enriched ßγ fraction; also, more ß(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched ßγ fraction. These results suggest that preferences for particular ßγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Marcaje Isotópico/métodos , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Aminoácidos/análisis , Técnicas de Cultivo de Célula , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/análisis , Células HEK293 , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Receptor de Adenosina A1/análisis , Receptor de Adenosina A2A/análisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Células Sf9RESUMEN
The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of ßγ-dimer of G-proteins (Gßγ) with ß-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gß (~36 kDa) was reduced, whereas the level of Gß localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.
Asunto(s)
Cilióforos/fisiología , Proteínas del Ojo/antagonistas & inhibidores , Reguladores de Proteínas de Unión al GTP/antagonistas & inhibidores , Fosfoproteínas/antagonistas & inhibidores , Cilióforos/citología , Cilióforos/efectos de la radiación , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Reguladores de Proteínas de Unión al GTP/genética , Reguladores de Proteínas de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Cinética , Luz , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Blepharisma japonicum ciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gbetagamma and the subsequent translocation of the Pdc-Gbetagamma complex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gbetagamma. However, as demonstrated by FRET-AP, Pdc-Gbetagamma complex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gbetagamma and beta-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (> or = 10 min). In contrast, an opposite effect, i.e. a transient decrease in the interaction between Gbetagamma and beta-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gbetagamma. In cells illuminated for a longer time (> or = 10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gbetagamma, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharisma cell body, which affects the interaction of Gbetagamma with beta-tubulin and cell cytoskeleton remodelling.
Asunto(s)
Cilióforos/efectos de la radiación , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Luz , Tubulina (Proteína)/metabolismo , Cilióforos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Ojo/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Reguladores de Proteínas de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/análisis , Isoquinolinas/farmacología , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Sulfonamidas/farmacología , Factores de Tiempo , Tubulina (Proteína)/análisisRESUMEN
Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit. Here, we show that co-expression of the Gbetagamma dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone beta reporter activity in LbetaT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gbeta1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gbetagamma did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gbetagamma co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gbetagamma subunits as transcriptional regulators.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Transcripción Genética , Animales , Línea Celular , Núcleo Celular/química , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/análisis , Humanos , Ratones , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Factor de Transcripción AP-1/metabolismoRESUMEN
The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Centrómero/química , Centrómero/metabolismo , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/efectos de los fármacos , Subunidades gamma de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/efectos de los fármacos , Ratones , Microtúbulos/química , Microtúbulos/efectos de los fármacos , Células 3T3 NIH , Nocodazol/farmacología , Células PC12 , Ratas , Tubulina (Proteína)/análisis , Moduladores de Tubulina/farmacologíaRESUMEN
We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCalpha) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Galphao, Gbeta, and PKCalpha were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Galphas, Gbeta, and PKCalpha in both groups, reduction in Galphai was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCalpha with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Grasas Insaturadas en la Dieta/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/análisis , Lípidos de la Membrana/análisis , Aceites de Plantas/administración & dosificación , Anciano , Anciano de 80 o más Años , Ésteres del Colesterol/análisis , Cromatografía de Gases , Diabetes Mellitus Tipo 2/fisiopatología , Membrana Eritrocítica/química , Ácidos Grasos/análisis , Ácidos Grasos Monoinsaturados/análisis , Femenino , Subunidades alfa de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/análisis , Proteínas de Unión al GTP/análisis , Humanos , Membrana Dobles de Lípidos/análisis , Masculino , Aceite de Oliva , Fosfolípidos/análisis , Proteína Quinasa C-alfa/análisis , Transducción de Señal/fisiologíaRESUMEN
The role of G protein-mediated signal transduction in the production of endolymph, an extracellular fluid of unusual ionic composition, is beginning to be understood. The identity of Galpha subunits in the stria vascularis and the spiral ligament of the lateral wall of the cochlear duct is well established. However, little is known about the presence of betagamma subunits. This study used immunohistochemistry to investigate the distribution of G protein betagamma subunits in the lateral wall of the cochlea. Temporal bones of 6- to 8-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The results show that the pattern of immunoreactivity varies for the G protein beta1-4 and gamma1-3, 5 and 7 subunits in the stria vascularis and spiral ligament. In the stria vascularis, immunoreactivity was detected for beta2, beta3, beta4, gamma1, gamma2 and gamma7 subunits. All five types of fibrocytes in the spiral ligament exhibited positive staining for gamma2 and gamma7. However, immunoreactivity for beta1-4 subunits was variable. Immunoreactivity for gamma3 and gamma5 subunits was not detected in the lateral cochlear wall. The expression pattern of G protein betagamma subunits in lateral wall provides a basis for interpreting the functions of G protein-coupled receptors in cochlear fluid homeostasis.
Asunto(s)
Cóclea/química , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/análisis , Animales , Femenino , Homeostasis , Inmunohistoquímica/métodos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gbeta (GFP-Gbeta) and GFP-Ggamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.
Asunto(s)
Dictyostelium/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Chaperonas Moleculares/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Membrana Celular/química , Citosol/química , Dictyostelium/genética , Dimerización , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/análisis , Subunidades gamma de la Proteína de Unión al GTP/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Chaperonas Moleculares/análisis , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Octoxinol , Polietilenglicoles/química , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
A cDNA clone encoding a novel G protein beta subunit of beta1 subclass, pfGbeta1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGbeta1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) pfGbeta1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein beta subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gbeta. The prediction of 3D structure of pfGbeta showed that pfGbeta belongs to beta-propeller family proteins whose members contain 4-8 antiparallel beta-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGbeta mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Gbetagamma and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Gbetagamma to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Gbetagamma increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGbetagamma that impaired the interactions of Gbetagamma with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Gbetagamma might interact with CaM and point to the important physiological function in modulating cellular functions.
Asunto(s)
Calmodulina/genética , Calmodulina/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Pinctada/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/fisiología , Calmodulina/química , Línea Celular , Clonación Molecular , ADN Complementario/biosíntesis , Subunidades beta de la Proteína de Unión al GTP/análisis , Humanos , Modelos Moleculares , Modelos Teóricos , Datos de Secuencia Molecular , Filogenia , Pinctada/química , Unión Proteica , ARN Mensajero/biosíntesis , Alineación de SecuenciaRESUMEN
By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.