An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

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.

Gpr85, a novel member of the G-protein coupled receptor family, prominently expressed in the developing mouse cerebral cortex.

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.

The brain-specific G-protein coupled receptor GPR85 with identical protein sequence in man and mouse maps to human chromosome 7q31.

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.

Competitive inhibition of human immunodeficiency virus type-1 protease by the Gag-Pol transframe protein.

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.