RESUMO
A synthetic version of the human D4 (hD4) dopamine receptor was prepared. The G/C content of the natural gene was reduced by 14% without altering the amino acid composition of the corresponding protein sequence. HEK293 cells were transfected with the synthetic hD4 gene and stable clones resistant to G418 selected. The hD4 receptor expressed from the synthetic gene had identical pharmacological characteristics to the native hD4 receptor [(1991) Nature 350, 610-619; (1992) Nature 358, 149-152]. Functional studies with cells expressing the synthetic hD4 gene indicated negative coupling of this receptor to adenylate cyclase.
Assuntos
Receptores de Dopamina D2 , Receptores Dopaminérgicos/biossíntese , Receptores Dopaminérgicos/metabolismo , Adenilil Ciclases/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Chlorocebus aethiops , Colforsina/farmacologia , DNA/química , DNA/metabolismo , Ergolinas/farmacologia , Genes Sintéticos , Humanos , Rim , Cinética , Dados de Sequência Molecular , Plasmídeos , Quimpirol , Receptores Dopaminérgicos/genética , Receptores de Dopamina D4 , Mapeamento por Restrição , Espiperona/metabolismo , Espiperona/farmacologia , TransfecçãoRESUMO
The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these 'orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of 'humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.
Assuntos
Desenho de Fármacos , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Humanos , Peptídeos/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Superfície Celular/efeitos dos fármacosRESUMO
It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores Purinérgicos P2 , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Ratos , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Purinérgicos P2Y , Homologia de Sequência de Aminoácidos , Uridina Difosfato Glucose/farmacologiaRESUMO
Using a genomics-based reverse pharmacological approach for screening orphan G-protein coupled receptors, we have identified and cloned a novel high-affinity histamine receptor. This receptor, termed AXOR35, is most closely related to the H3 histamine receptor, sharing 37% protein sequence identity. A multiple responsive element/cyclic AMP-responsive element-luciferase reporter assay was used to identify histamine as a ligand for AXOR35. When transfected into human embryonic kidney 293 cells, the AXOR35 receptor showed a strong, dose-dependent calcium mobilization response to histamine and H3 receptor agonists including imetit and immepip. Radioligand binding confirmed that the AXOR35 receptor was a high-affinity histamine receptor. The pharmacology of the AXOR35 receptor was found to closely resemble that of the H3 receptor; the major difference was that (R)-alpha-methylhistamine was a low potency agonist of the AXOR35 receptor. Thioperamide is an antagonist at AXOR 35. Expression of AXOR35 mRNA in human tissues is highest in peripheral blood mononuclear cells and in tissues likely to contain high concentrations of blood cells, such as bone marrow and lung. In situ hybridization analysis of a wide survey of mouse tissues showed that mouse AXOR35 mRNA is selectively expressed in hippocampus. The identification and localization of this new histamine receptor will expand our understanding of the physiological and pathological roles of histamine and may provide additional opportunities for pharmacological modification of these actions.
Assuntos
Histamina/metabolismo , Receptores Histamínicos/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Clonagem Molecular , Relação Dose-Resposta a Droga , Expressão Gênica , Genes Reporter , Humanos , Luciferases , Camundongos , Dados de Sequência Molecular , Ensaio Radioligante , Receptores Histamínicos/metabolismo , Receptores Histamínicos H3/química , Receptores Histamínicos H3/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , TrítioRESUMO
The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.
Assuntos
Proteínas de Membrana , Receptores de Leucotrienos/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Clonagem Molecular , Humanos , Leucotrieno D4/farmacologia , Dados de Sequência Molecular , Toxina Pertussis , Receptores de Leucotrienos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.