RESUMO
Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.
Assuntos
Doenças do Gato/metabolismo , Neoplasias da Íris/veterinária , Melanoma/veterinária , Animais , Gatos , Feminino , Imunofluorescência/veterinária , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/biossíntese , Neoplasias da Íris/metabolismo , Melanoma/metabolismo , Biossíntese de Proteínas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Supressoras de Tumor/biossínteseRESUMO
Gαq, the α-subunit of Gq protein, is ubiquitously expressed in mammalian cells. It initially attracted attention for its physiological significance in cardiovascular system. In recent years, studies have also indicated the important roles of Gαq in regulating immunity, supplying us a new insight into the mechanism of immune regulation. T helper type 17 (Th17) cells are potent inducers of tissue inflammation. Many studies have shown that Th17 cells are major effector cells in the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions such as rheumatoid arthritis (RA). One of our previous studies has shown that Gαq negatively controls the disease activity of RA. However, how Gαq controls the pathogenesis of autoimmune disease is not clear. Whether this effect is via the regulation of Th17 differentiation is still not known. We aimed to find out the role of Gαq in control of Th17 differentiation. We investigated the relationship between Gαq and Th17 in RA patients. We then investigated the mechanism of how Gαq regulated Th17 differentiation by using Gnaq(-/-) mice. We observed that the expression of Gαq was negatively associated with interleukin-17A expression in RA patients, indicating that Gαq negatively controlled the differentiation of Th17 cells. By using Gnaq(-/-) mice, we demonstrated that Gαq inhibited the differentiation of Th17 cell via regulating the activity of extracellular signal-regulated kinase-1/2 to control the expression of STAT3 (signal transducer and activator of transcription 3) and RORα (RAR-related orphan receptor-α). These data suggest the possibility of targeting Gαq to develop a novel therapeutic regimen for autoimmune disease.
Assuntos
Artrite Reumatoide/patologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Células Th17/patologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Flavonoides/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Regulação da Expressão Gênica , Humanos , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Linfopoese/genética , Linfopoese/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Quimera por Radiação , Fator de Transcrição STAT3/fisiologia , Espondilite Anquilosante/genética , Espondilite Anquilosante/metabolismo , Células Th17/imunologiaRESUMO
G protein-coupled receptors are the most pervasive signaling superfamily in the body and act as receptors to endogenous agonists and drugs. For ß-agonist-mediated bronchodilation, the receptor-G protein-effector network consists of the ß2-adrenergic receptor (ß2AR), Gs, and adenylyl cyclase, expressed on airway smooth muscle (ASM). Using ASM-targeted transgenesis, we previously explored which of these three early signaling elements represents a limiting factor, or bottleneck, in transmission of the signal from agonist binding to ASM relaxation. Here we overexpressed Gαs in transgenic mice and found that agonist-promoted relaxation of airways was enhanced in direct proportion to the level of Gαs expression. Contraction of ASM from acetylcholine was not affected in Gαs transgenic mice, nor was relaxation by bitter taste receptors. Furthermore, agonist-promoted (but not basal) cAMP production in ASM cells from Gαs-transgenic mice was enhanced compared with ASM from nontransgenic littermates. Agonist-promoted inhibition of platelet-derived growth factor-stimulated ASM proliferation was also enhanced in Gαs mouse ASM. The enhanced maximal ß-agonist response was of similar magnitude for relaxation, cAMP production, and growth inhibition. Taken together, it appears that a limiting factor in ß-agonist responsiveness in ASM is the expression level of Gαs. Gene therapy or pharmacological means of increasing Gαs (or its coupling efficiency to ß2AR) thus represent an interface for development of novel therapeutic agents for improvement of ß-agonist therapy.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratório/metabolismo , Transdução de Sinais , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Broncodilatadores/farmacologia , Linhagem Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Camundongos Transgênicos , Músculo Liso/patologia , Receptores Adrenérgicos beta 2/genética , Sistema Respiratório/patologiaRESUMO
In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The hormonal action of GSS on follicle cells involves its receptor, G-proteins and adenylyl cyclase. However, GSS failed to induce 1-MeAde and cAMP production in follicle cells of ovaries during oogenesis. At the maturation stage, follicle cells acquired the potential to respond to GSS by producing 1-MeAde and cAMP. Adenylyl cyclase activity in follicle cells of fully grown stage ovaries was also stimulated by GSS in the presence of GTP. These activations depended on the size of oocytes in ovaries. The α subunit of Gs-proteins was not detected immunologically in follicle cells of oogenesis stage ovaries, although Gαi and Gαq were detectable. Using specific primers for Gαs and Gαi, expression levels of Gαs in follicle cells were found to increase significantly as the size of oocytes in ovaries increased, whereas the mRNA levels of Gαi were almost constant regardless of oocyte size. These findings strongly suggest the potential of follicle cells to respond to GSS by producing 1-MeAde and cAMP is brought by de novo synthesis of Gαs-proteins.
Assuntos
Adenina/análogos & derivados , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Hormônios de Invertebrado/metabolismo , Neuropeptídeos/metabolismo , Folículo Ovariano/metabolismo , Estrelas-do-Mar/metabolismo , Adenina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Feminino , Hormônios de Invertebrado/farmacologia , Neuropeptídeos/farmacologia , Folículo Ovariano/efeitos dos fármacosRESUMO
Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Camundongos , Subunidades Proteicas , Transdução de SinaisRESUMO
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 Gα(i), Gα(q), and Gα(13) proteins to <5% of those of wild-type cells. The amounts of Gα(s) and total Gß protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of Gα(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein α 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 Gα(i) and Gα(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gßγ. Finally, Gα(i), Gα(q), and Gß(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 Gα subunits with cellular membranes.
Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Membrana Celular/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/genéticaRESUMO
BACKGROUND: Pharmacologic and ablative therapies for atrial fibrillation (AF) have suboptimal efficacy. Newer gene-based approaches that target specific mechanisms underlying AF are likely to be more efficacious in treating AF. Parasympathetic signaling appears to be an important contributor to AF substrate. OBJECTIVE: The purpose of this study was to develop a nonviral gene-based strategy to selectively inhibit vagal signaling in the left atrium and thereby suppress vagal-induced AF. METHODS: In eight dogs, plasmid DNA vectors (minigenes) expressing Gα(i) C-terminal peptide (Gα(i)ctp) was injected in the posterior left atrium either alone or in combination with minigene expressing Gα(o)ctp, followed by electroporation. In five control dogs, minigene expressing scrambled peptide (Gα(R)ctp) was injected. Vagal- and carbachol-induced left atrial effective refractory periods (ERPs), AF inducibility, and Gα(i/o)ctp expression were assessed 3 days following minigene delivery. RESULTS: Vagal stimulation- and carbachol-induced effective refractory period shortening and AF inducibility were significantly attenuated in atria receiving a Gα(i2)ctp-expressing minigene and were nearly eliminated in atria receiving both Gα(i2)ctp- and Gα(o1)ctp-expressing minigenes. CONCLUSION: Inhibition of both G(i) and G(o) proteins is necessary to abrogate vagal-induced AF in the left atrium and can be achieved via constitutive expression of Gα(i/o)ctps expressed by nonviral plasmid vectors delivered to the posterior left atrium.
Assuntos
Fibrilação Atrial/terapia , Carbacol/farmacologia , DNA/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Terapia Genética/métodos , Átrios do Coração/inervação , Nervo Vago/fisiopatologia , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Agonistas Colinérgicos/farmacologia , Cães , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/efeitos dos fármacos , Expressão Gênica , Vetores Genéticos/farmacologia , Átrios do Coração/fisiopatologia , Nervo Vago/efeitos dos fármacosRESUMO
Most terrestrial vertebrates possess anatomically distinct olfactory organs: the olfactory epithelium (OE) and the vomeronasal organ (VNO). In rodents, olfactory receptors coupled to Gα(olf) are expressed in the OE, whereas vomeronasal receptors type 1 (V1R) and vomeronasal receptors type 2 (V2R), coupled to Gα(i2) and Gα(o) , respectively, are expressed in the VNO. These receptors and G proteins are thought to play important roles in olfactory perception. However, we previously reported that only V2R and Gα(o) expression is detected in the Xenopus laevis VNO. As X. laevis spends its entire life in water, we considered that expression of limited types of chemosensory machinery in the VNO might be due to adaptation of the VNO to aquatic life. Thus, we analyzed the expression of G proteins in the VNO and the accessory olfactory bulb (AOB) of the adult Japanese toad, Bufo japonicus, because this species is well adapted to a terrestrial life. By using immunohistochemical analysis in combination with in situ hybridization and DiI labeling, we found that B. japonicus Gα(olf) and Gα(o) were expressed in the apical and middle-to-basal layer of the vomeronasal neuroepithelium, and that the axons of these Gα(olf) - and Gα(o) -expressing vomeronasal neurons projected to the rostral and caudal accessory olfactory bulb, respectively. These results strongly suggest that both the Gα(olf) - and Gα(o) -mediated signal transduction pathways function in the B. japonicus VNO. The expression of Gα(olf) in the B. japonicus VNO may correlate with the detection of airborne chemical cues and with a terrestrial life.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Neurônios Receptores Olfatórios/metabolismo , Órgão Vomeronasal/metabolismo , Animais , Western Blotting , Bufonidae , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/análise , Imuno-Histoquímica , Hibridização In Situ , Masculino , Transdução de Sinais/fisiologiaRESUMO
The recent identification of Gα(z) expression in C2C12 myoblasts and its demonstrated interaction with the transcription factor Eya2 inferred an unanticipated role of Gα(z) in muscle development. In the present study, endogenous Gα(z) mRNA and protein expressions in C2C12 cells increased upon commencement of myogenesis and peaked at around 4-6days after induction but were undetectable in adult skeletal muscle. Surprisingly, stable expression of recombinant Gα(z) in C2C12 myoblasts strongly suppressed myotube formation upon serum deprivation, and the constitutively active mutant Gα(z)QL exerted more pronounced effects. Transcriptional activities of reporter genes responsive to early (MyoD, MEF2 and myogenin) and late (muscle creatine kinase and myosin heavy chain) myogenic markers were reduced by transiently expressed Gα(z)QL. Membrane attachment of Gα(z) was apparently required for the suppressive effects because a fatty acylation-deficient Gα(z) mutant could not inhibit myogenin expression. Introduction of siRNA against Gα(z) enhanced myogenin-driven luciferase activity and increased myosin heavy chain expression. Immunostaining of C2C12 cells over-expressing Gα(z) showed delayed nuclear expression of myogenin and severe myotube deformation. Gα(z) expression was accompanied by reduced levels of Rock2, RhoA and RhoGAP, enhanced expression of Rnd3, and a reduction of serum-responsive factor-driven reporter activity. These results support a novel role of Gα(z) in restraining myogenic differentiation through the disruption of Rho signaling.
Assuntos
Diferenciação Celular , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Mioblastos Esqueléticos/fisiologia , Animais , Linhagem Celular , Creatina Quinase Forma MM/biossíntese , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Miogenina/fisiologia , Cadeias Pesadas de Miosina/biossíntese , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Glutationa Transferase/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Animais , Baculoviridae/genética , Ativação Enzimática , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Expressão Gênica , Glutationa Transferase/biossíntese , Glutationa Transferase/química , Glutationa Transferase/genética , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , SpodopteraRESUMO
It has been shown that the disruption of the alpha-subunit gene of heterotorimeric G-proteins (Galpha) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Galpha in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Galpha gene were detected in the all d1 alleles. By western blot, the Galpha proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Galpha protein was occurred and even in this case the Galpha protein was only just detectable in the plasma membrane fraction. Given that the Galpha protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Galpha protein, it is likely that a proper conformation of the Galpha is necessary for accumulation of Galpha protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL.
Assuntos
Alelos , Membrana Celular/enzimologia , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Oryza/enzimologia , Proteínas de Plantas/biossíntese , Brassinosteroides , Membrana Celular/genética , Colestanóis/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Oryza/genética , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esteroides Heterocíclicos/farmacologiaRESUMO
Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Neuropeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Sequência de Aminoácidos , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/biossíntese , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/biossíntese , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Morfina/antagonistas & inibidores , Medição da Dor , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.
Assuntos
Membrana Celular/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Dimerização , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades beta da Proteína de Ligação ao GTP/biossíntese , Subunidades gama da Proteína de Ligação ao GTP/biossíntese , Modelos Biológicos , Dobramento de Proteína , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.
Assuntos
Aplysia/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Receptores de Inositol 1,4,5-Trifosfato/genética , Órgãos dos Sentidos/enzimologia , Olfato/genética , Fosfolipases Tipo C/genética , Sequência de Aminoácidos , Animais , Aplysia/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Microdissecção , Dados de Sequência Molecular , Feromônios/fisiologia , Órgãos dos Sentidos/fisiologia , Transdução de Sinais/genética , Fosfolipases Tipo C/biossínteseRESUMO
GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.
Assuntos
Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Sequência de Bases , Encéfalo/citologia , Linhagem Celular , AMP Cíclico/biossíntese , Chaperona BiP do Retículo Endoplasmático , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both Galpha and Gbetagamma subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both Gbetagamma expression and subsequent signaling by chaperoning nascent Gbeta and facilitating heterodimer formation with Ggamma subunits (Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. (2005) EMBO J. 24, 1965-1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. (2005) J. Biol. Chem. 280, 20042-20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the Ggamma subunit but not Gbeta or Galpha subunits. Furthermore, we demonstrate that DRiP78 and the Gbeta subunit can compete for the Ggamma subunit. DRiP78 also protects Ggamma from degradation until a stable partner such as Gbeta is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific Gbetagamma heterodimers, as selectivity was observed among Ggamma isoforms for interaction with DRiP78 depending on the presence of particular Gbeta subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of Gbetagamma by linking Ggamma to PhLP.Gbeta complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of Gbetagamma subunits of the G protein.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/biossíntese , Subunidades gama da Proteína de Ligação ao GTP/biossíntese , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas Fetais , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica/fisiologia , Transporte Proteico/fisiologiaRESUMO
Opiate dependence develops due to changes in intracellular signaling caused by long-term exposure to morphine. Here we investigated changes in the mRNA expression of the main classes of G-protein alpha (Galpha) subunits in various brain regions in morphine-dependent rats. Rats received increasing doses of morphine, 10-50 mg/kg, b.i.d., for 14 days. G-protein alpha-subunit mRNA expression was determined shortly following the conclusion of chronic morphine administration (2 h after the final dose) and during withdrawal (48 h after the final dose). Significant changes in mRNA expression for Galpha subunits were observed in several brain areas during withdrawal, while the changes were much less evident or absent 2 h after the final drug injection. Changes in mRNA expression were particularly evident in the nucleus accumbens (increases in Galpha(12), Galpha(q), Galpha(11), and Galpha(o) during withdrawal, increase in Galpha(i) and decrease in Galpha(s) just following treatment). The direction of the changes, which were not all significant, for Galpha(12), Galpha(q), and Galpha(11) was generally consistent in the amygdala and prefrontal cortex; changes in G proteins coupled to the adenylyl cyclase cascade were less consistent. These results suggest that morphine dependence leads to alterations in intracellular signaling, which are reflected in changes in the expression of genes encoding various G proteins. The results may explain why signs of opiate dependence are not expressed during chronic administration of morphine, but only after cessation of the treatment.
Assuntos
Comportamento Animal/efeitos dos fármacos , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Dependência de Morfina , Morfina , RNA Mensageiro/biossíntese , Síndrome de Abstinência a Substâncias , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Injeções Intraperitoneais , Masculino , Morfina/administração & dosagem , Morfina/efeitos adversos , Dependência de Morfina/metabolismo , Dependência de Morfina/fisiopatologia , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Wistar , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia , Fatores de TempoRESUMO
Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.
Assuntos
Proteínas Fúngicas/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Mucor/genética , Proteínas Recombinantes/biossíntese , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Dados de Sequência Molecular , Mucor/metabolismo , Filogenia , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Rodopsina/metabolismo , Transdução de Sinais/fisiologia , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Ratos , Deleção de Sequência/genética , Transdução de Sinais/genéticaRESUMO
Genetics and proteomics determine structure and function of normal tissues, and the molecular alterations that underlie tumorigenesis result in changes in these aspects of tissue biology in neoplasms. We review the known genetic alterations in pituitary tumors. These include the oncogenic Gsalpha protein (GSP)-activating mutations, and pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4), as well as tumor suppressor gene mutations associated with multiple endocrine neoplasia type 1 (MEN1). Other candidates identified from expression profiling include pituitary tumor-transforming gene (PTTG), GADD45, and bone morphogenic protein (BMP)4. Proteomic changes in pituitary tumors include classical alterations identified by immunohistochemistry as well as epigenetic reductions in p27. The underlying mechanisms for dysregulated cell adhesive molecules including cadherins and FGFRs are reviewed. The combined use of genetic and proteomic approaches will enhance novel drug therapeutic development.