RESUMO
Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein-protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1-TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1-TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , RatosRESUMO
A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptores de Somatostatina/fisiologia , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Somatostatina/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Bioensaio , Divisão Celular , Membrana Celular/metabolismo , Primers do DNA/química , Dados de Sequência Molecular , Feromônios/fisiologia , Ratos , Receptores de Fator de Acasalamento , Receptores de Peptídeos/fisiologia , Proteínas Recombinantes , Saccharomyces cerevisiae/crescimento & desenvolvimento , Somatostatina/químicaRESUMO
A new superfamily of K+ channels has emerged in the past 2 years. Notable for possessing two pore-forming P domains in each subunit, members of the superfamily have been recognized through phylogeny from micro-organisms to humans. Four subfamilies of two P domain channels have been isolated thus far; among these are the first cloned examples of outward rectifier and open rectifier (or leak) K+ channels. The two P domain K+ channels offer a new perspective from which to glimpse the molecular basis for function and dysfunction of K+-selective ion channels.
Assuntos
Canais de Potássio/química , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
G-protein-coupled receptors are an important class of therapeutic drug targets by virtue of their roles in the regulation of diverse cellular functions. Recent advances in the expression of heterologous G-protein-coupled receptors in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of their ligand-induced activation. Implementation of this new technology in the high-throughput screening of compound libraries has enabled the discovery of novel ligands for the G-protein-coupled somatostatin receptor. This article describes the broad applicability of the technology and its use in drug discovery.
Assuntos
Indústria Farmacêutica/tendências , Proteínas de Ligação ao GTP , Receptores de Somatostatina , Saccharomyces cerevisiae/químicaRESUMO
The presence of alpha2-adrenoceptors in membranes from omental and s.c. adipose tissue from gilts and barrows was shown in saturation binding assays with [3H]yohimbine. Four trials tested effects of alpha2-adrenoceptor antagonists (A2AA) on plasma concentrations of NEFA and urea nitrogen (PUN). In Trial 1, barrows were given i.v. injections of saline, 200 microg/kg BW of one of three A2AA (efaroxan, idazoxan, or RX821002), or 25 microg/kg BW of isoproterenol. Concentrations of NEFA were measured in plasma harvested every 15 min from 1 h before to 2 h after treatment. Compared with results for saline-treated pigs, areas under the curve (AUC) for NEFA were increased (P < .05) by efaroxan, RX821002, and isoproterenol. In Trial 2, barrows received i.v. doses of saline, efaroxan (200 or 400 microg/kg BW), or RX821002 (200 or 400 microg/kg BW). Levels of NEFA were quantified in plasma obtained at 15-min intervals through 2 h after treatment. Among pigs treated with RX821002 at 400 microg/kg BW, mean NEFA AUC was more than three times greater (P < .05) than that for saline-treated animals. Trial 3 tested whether NEFA responses to A2AA were due to direct effects on alpha2-receptors or involved beta-adrenoceptor mediation. Pigs were first treated i.v. with saline or propranolol (1 mg/kg BW). One hour later, pigs were treated i.v. with RX821002 (400 microg/kg BW) or the beta-adrenoceptor agonist cimaterol (25 microg/kg BW). Compared to values for pigs treated with saline at both injections, NEFA AUC among pigs treated with saline at the first injection and RX821002 at the second doubled (P > .05). Plasma NEFA AUC among pigs treated with saline then cimaterol rose nearly fourfold (P < .05) compared with saline-treated controls. Mean NEFA AUC among propranolol-treated pigs was similar to values for saline-treated pigs, suggesting beta-adrenoceptor involvement in the effect of A2AA on NEFA. In Trial 4, pigs were treated s.c. 10 times at 8-h intervals with saline, RX821002 (400 microg/[kg BW x injection]), cimaterol (20 microg/[kg BW x injection]) or recombinant porcine somatotropin (rpST; 1 mg/[pig-injection]). After the 10th treatment, only cimaterol increased NEFA AUC compared to saline-treated controls (P < .05). Mean PUN AUC was reduced by RX821002 and rpST compared to controls; PUN among rpST-treated pigs was lower than that among RX821002-treated pigs (P < .05). In summary, A2AA increase lipolysis in swine by potentiating lipolytic effects of endogenous catecholamines on beta-adrenoceptors. Reduced PUN suggests improved nitrogen efficiency may result from treatment with A2AA.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Nitrogênio da Ureia Sanguínea , Ácidos Graxos não Esterificados/sangue , Suínos/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Área Sob a Curva , Benzofuranos/farmacologia , Feminino , Idazoxano/análogos & derivados , Idazoxano/farmacologia , Imidazóis/farmacologia , Isoproterenol/farmacologia , Masculino , Distribuição AleatóriaRESUMO
We studied the effects of alpha2-adrenoceptor antagonists (A2AA) on nitrogen (N) partitioning. The diets fed contained 19.8% CP and 1.15% lysine. Pigs were fed the diet as a percentage of BW equaling approximately 90% of voluntary intake. In Trial 1, pigs (n = 11/treatment) were fed a basal diet and injected s.c. at 8-h intervals for 11 d with saline, RX821002 (25 mg/injection), or cimaterol (.6 mg/injection). Compared to saline-treated pigs, urinary N, as a percentage of N eaten, decreased among pigs injected with RX821002 (15%, P < .05) or cimaterol (17%, P < .05). In Trial 2, pigs got saline (n = 6) or 25 mg RX821002 (n = 6) as s.c. injections three times daily, or they were fed a diet containing 150 ppm RX821002 and injected thrice daily with saline (n = 6) for 11 d. The RX821002 lowered apparent DM and N digestibility (P < .05). Compared to controls, RX821002 lowered urinary N, as a percentage of N eaten, 15 and 18% when given by injections or per os, respectively, but effects were not significant. Trial 3 evaluated the effects of RX821002 fed at levels of 0 (n = 6), 37.5 (n = 5), 75 (n = 6), or 150 ppm (n = 6). Contrasts showed linear dose-dependent decreases in gain and apparent N digestibility (P < .05). Compared to untreated controls, urinary N, expressed as a percentage of N consumed, decreased 2, 12, and 10% among pigs fed diets with 37.5, 75, or 150 ppm RX821002, respectively, but effects were not significant. Trial 4 compared N balance in pigs (n = 6/treatment) fed basal diet or diet with 100 ppm RX821002 to that of pigs fed diets with 25 or 100 ppm yohimbine. Treatments reduced apparent N and DM digestibility (P < .05). Urinary N, as a percentage of N consumed, decreased 16 (P > .05), 18 (P < .05), and 24% (P < .05) for 100 ppm RX821002, 25 ppm yohimbine, or 100 ppm yohimbine, respectively. Data from Trials 2, 3, and 4 from control pigs (n = 18) or pigs fed A2AA (all A2AA sources and doses; n = 41) were pooled and analyzed. Feeding A2AA decreased apparent N and DM digestibility (P < .01). The fact that fecal moisture content was higher in pigs fed A2AA suggests rate of digesta passage increased and offers an explanation for reduced N and DM digestibility in treated pigs. Despite adverse effects of A2AA, efficiency of postabsorptive N metabolism increased. As a percentage of N consumed and compared to control pigs, urinary N decreased 15% (P < .01) and retained N increased 12% (P < .05) in animals fed A2AA. Data from these studies show net efficiency of N metabolism is improved in swine given A2AA.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Idazoxano/análogos & derivados , Nitrogênio/metabolismo , Suínos/metabolismo , Administração Oral , Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/administração & dosagem , Agonistas Adrenérgicos beta/farmacologia , Ração Animal , Animais , Digestão/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanolaminas/administração & dosagem , Etanolaminas/farmacologia , Idazoxano/administração & dosagem , Idazoxano/farmacologia , Injeções Subcutâneas/veterinária , Modelos Lineares , Masculino , Nitrogênio/urina , Distribuição Aleatória , Ioimbina/administração & dosagem , Ioimbina/farmacologiaRESUMO
BACKGROUND: This study investigated the progression and clinical relevance of biochemical resorption marker values during fracture healing in osteoporosis. PATIENTS AND METHODS: In 44 patients with distal radius fractures and 29 patients without fractures, the blood and urine concentrations of pyridinoline (PYD), deoxypyridinoline (DPD), N-telopeptides (NTx), and bone sialoprotein (BSP) were recorded on the day of trauma as well as during further progression. All postmenopausal patients underwent bone density measurement. Accordingly, patients were divided into premenopausal, postmenopausal osteoporotic, and postmenopausal nonosteoporotic groups. RESULTS: Between the groups, PYD, DPD, and NTx showed significant differences in their initial values. However, their further relative progression was primarily affected by the chosen therapy. CONCLUSION: Bone resorption markers can diagnostically point to osteoporosis and are significant parameters in fracture healing.
Assuntos
Biomarcadores/metabolismo , Reabsorção Óssea/fisiopatologia , Consolidação da Fratura/fisiologia , Fraturas Espontâneas/fisiopatologia , Osteoporose Pós-Menopausa/fisiopatologia , Fraturas do Rádio/fisiopatologia , Traumatismos do Punho/fisiopatologia , Absorciometria de Fóton , Adulto , Idoso , Aminoácidos/metabolismo , Densidade Óssea/fisiologia , Colágeno Tipo I/metabolismo , Feminino , Fraturas Espontâneas/cirurgia , Humanos , Sialoproteína de Ligação à Integrina , Pessoa de Meia-Idade , Peptídeos/metabolismo , Valores de Referência , Sialoglicoproteínas/metabolismo , Traumatismos do Punho/cirurgiaRESUMO
G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein alpha subunit are necessary for membrane attachment. Here we identify a region within the yeast G alpha (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsalpha with the corresponding sequence from Gpa1. Gsalpha is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsaplha chimeras were assayed for proper membrane localization by functional complementation of a gpa1Delta ;) mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas Fúngicas/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/química , Teste de Complementação Genética , Estrutura Terciária de Proteína , Ratos , Receptores Purinérgicos P1/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
We purified a Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over-production (11-fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A-CMK1 fusion protein, possessed significant residual Ca2+/CAM-dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM-dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Clonagem Molecular , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligopeptídeos/química , Mapeamento por RestriçãoRESUMO
The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.
Assuntos
Receptores Muscarínicos/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Western Blotting , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/genética , Humanos , Ligantes , Dados de Sequência Molecular , Plasmídeos/genética , Ensaio Radioligante , Receptor Muscarínico M1 , Receptor Muscarínico M3 , Receptor Muscarínico M5 , Receptores Muscarínicos/genéticaRESUMO
The rat A2a adenosine receptor, a G protein-coupled receptor, was functionally expressed in the yeast Saccharomyces cerevisiae. High affinity binding sites for A2a adenosine agonists were detected in yeast membranes containing the endogenous Grx protein Gpa1. Agonist saturation binding isotherms using [3H]5'-N-ethylcarboxamidoadenosine indicated that the A2a adenosine receptor expressed in yeast cell membranes displays pharmacological properties equivalent to those observed when the receptor is expressed in human embryonic kidney 293 cell membranes. The rank order of potency of various agonists in [3H]5'-N-ethylcarboxamidoadenosine competition binding assays performed with yeast cell membranes was comparable to that seen for the receptor expressed in mammalian cell membranes. Adenosine agonist-dependent growth response of yeast strains expressing the A2a adenosine receptor was elicited via activation of the yeast pheromone-response pathway. Induction of a pheromone-responsive FUS1-HIS3 reporter gene in far1 his3 cells permits cell growth in medium lacking histidine. The sensitivity of the bioassay was increased by deletion of the STE2 gene, which encodes the yeast alpha-mating pheromone receptor. The growth response was dose dependent, and agonists of varying affinities displayed a rank order of potency comparable to that observed in competition binding assays. Agonist-activated growth assays performed in liquid culture gave ED50 values for various adenosine agonists consistent with reported Kd alpha values. Yeast strains expressing a single receptor/G protein complex will be useful as a model system for the study of receptor/G protein interactions in vivo.
Assuntos
Feromônios/fisiologia , Receptores Purinérgicos P1/fisiologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida) , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Humanos , Rim/fisiologia , Cinética , Fenetilaminas/metabolismo , Fenetilaminas/farmacologia , Feromônios/metabolismo , Agonistas do Receptor Purinérgico P1 , Ratos , Receptores Purinérgicos P1/metabolismo , Receptores de Somatostatina/metabolismo , Receptores de Somatostatina/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TrítioRESUMO
A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the "leak" conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Bário/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Proteínas de Drosophila , Drosophila melanogaster , Feminino , Teste de Complementação Genética , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos/fisiologia , Proteínas de Plantas , Potássio/metabolismo , Canais de Potássio/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas RGS/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Retroalimentação , Sistema de Sinalização das MAP Quinases , Dados de Sequência Molecular , FosforilaçãoRESUMO
The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Primase , Nucleotídeos de Desoxiadenina/farmacologia , Oligonucleotídeos/metabolismo , Poli T/metabolismo , RNA Ligase (ATP)/metabolismo , Moldes Genéticos , Fatores de TempoRESUMO
Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent G alpha s stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast G alpha protein with proteins containing C-terminal regions of G alpha s. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH29 and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP , Receptores de Neuropeptídeos/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Feromônios/metabolismo , Plasmídeos/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Transdução de SinaisRESUMO
To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.
Assuntos
Arginina Vasopressina/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Vasopressinas/química , Receptores de Vasopressinas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Bovinos , Membrana Celular/fisiologia , Clonagem Molecular , Primers do DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Biblioteca Gênica , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Subunidades Proteicas , Receptores de Vasopressinas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
The study of the five somatostatin receptor subtypes (SSTx, where x is the subtype number) has been hampered by the lack of high affinity antagonists. Potent and selective antagonists would increase our understanding of SST structure, function, and regulation. In this study, the identification of novel disulfide-linked cyclic octapeptide antagonists of somatostatin is described. The antagonists contain a core structure of a DL-cysteine pair at positions 2 and 7 of the peptides. Substitution of a D-cysteine at position 2 with an L-cysteine converts the full antagonist into a full agonist. All somatostatin receptor subtypes are coupled to inhibition of adenylate cyclase. The functional properties of these peptides have been determined in radioligand binding assays, in functional coupling of the SST2 subtype to yeast pheromone response pathway, and in cAMP accumulations. One peptide antagonist [Ac-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2] displays a binding affinity to SST2 comparable with that observed for the native hormone (Ki = 0.2 nM) and reverses somatostatin-mediated inhibition of cAMP accumulation in rat somatomammotroph GH4C1 cells, cells transfected with the SST2 and SST5 subtypes, as well as somatostatin-stimulated growth of yeast cells expressing the SST2 subtype. This class of somatostatin antagonists, which are the first to be described, should be useful for determination of somatostatin's diverse functions in vivo and in vitro.
Assuntos
Somatostatina/análogos & derivados , Somatostatina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Ligação Competitiva , AMP Cíclico/metabolismo , Radioisótopos do Iodo , Peptídeos/metabolismo , Peptídeos/farmacologia , Ensaio Radioligante , Ratos , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Somatostatin (SRIF) is the main inhibitory peptide regulating growth hormone (GH) secretion. It has been difficult to establish the role of endogenous SRIF release in the absence of pure SRIF antagonists. Although several SRIF antagonists have recently been described, none have been shown to possess in vivo activity in the absence of added SRIF. Here, an SRIF antagonist with no detectable agonist activity has been identified from a synthetic combinatorial hexapeptide library containing 6.4 x 10(7) unique peptides. Each peptide in the library is amino-terminally acetylated and carboxyl-terminally amidated and consists entirely of D-amino acids. A SRIF-responsive yeast growth assay was used as a primary screening tool, and cAMP accumulation, competitive binding, and microphysiometry also were used to confirm and further characterize SRIF antagonist activity. The hexapeptide library was screened in stepwise iterative fashion to identify AC-178,335, a pure SRIF antagonist of the sequence Ac-hfirwf-NH2. This D-hexapeptide bound SRIF receptor type 2 with an affinity constant (Ki) of 172 +/- 12 nM, blocked SRIF inhibition of adenylate cyclase in vitro (IC50 = 5.1 +/- 1.4 microM), and induced GH release when given alone (50 micrograms intravenously) to anesthetized rats with or without pretreatment with a long-acting SRIF agonist.