RESUMO
We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.
Assuntos
Clonagem Molecular/métodos , Etiquetas de Sequências Expressas , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 3/genética , Sequência Conservada , Bases de Dados como Assunto , Éxons/genética , Perfilação da Expressão Gênica , Humanos , Íntrons/genética , Ligantes , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Filogenia , Mapeamento Físico do Cromossomo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Superfície Celular/química , Receptores Acoplados a Proteínas G/química , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Alinhamento de Sequência , Uridina Difosfato Glucose/metabolismoRESUMO
The G-protein coupled receptors (GPCRs) characterized by seven transmembrane domains represent the largest receptor superfamily to date and are implied in diverse cell signaling events, its members being present in a diversity of organs and tissues. Here we report the expression of Gpr85, a novel member of this gene family during mouse embryonal development and in the adult brain. Transcripts of Gpr85 were detected predominantly in tissues of neuroectodermal origin. In the central nervous system Gpr85 was expressed during phases of early neuronal differentiation. Highest transcript levels were observed in the developing cerebral cortex, pointing to a specific function of this gene for differentiation processes in the cerebral cortex. In addition, expression was also detected in derivatives of the neural crest and developing teeth.
RESUMO
A new G-protein coupled receptor, GPR85, was identified in man and mouse, which is completely conserved at the amino acid level. Transcripts of gpr85 were found in several human brain regions and, at lower levels, in spleen and placenta, whereas in mouse, gpr85 is confined to the brain. The hgpr85 gene was localized to human chromosome 7q31 by the polymerase chain reaction utilizing a radiation hybrid panel.
Assuntos
Química Encefálica , Cromossomos Humanos Par 7 , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Etiquetas de Sequências Expressas , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.
