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GPR99, a new G protein-coupled receptor with homology to
a new subgroup of nucleotide receptors捕捉青蛙
Timo Wittenberger1*,Susanne Hellebrand1*,Antonia Munck1,Hans-Jürgen Kreienkamp2,H. Chica Schaller1,and Wolfgang Hampe1+
1Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistr. 52, D-20246 Hamburg, Germany
2Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistr.52, D-20246 Hamburg, Germany
合同样本
* The authors contributed equally to the work.
+Correspondence: Dr. W. Hampe
Institut für Medizinische Biochemie und Molekularbiologie
Abteilung für Molekulare Zellbiologie
52
Martinistr.
D-20246 Hamburg, Germany
Tel.:
+49-40-42803-9967
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Fax:
e-mail: hampe@uke.uni-hamburg.de流通是什么意思
Abstract
Background
Bad on quence similarity, the superfamily of G protein-coupled receptors (GPRs) can be subdivided into veral subfamilies, the members of which often share similar ligands. The quence data provided by the human genome project allows to identify new GPRs by in silico homo
logy screening and to predict their ligands.
Results
By arching the human genomic databa with known nucleotide receptors we discovered the gene for GPR99, a new orphan GPR. The mRNA of GPR99 was found in kidney and placenta. Phylogenetic analysis groups GPR99 into the P2Y subfamily of GPRs. Bad on the phylogenetic tree we propo a new classification of P2Y nucleotide receptors into two subgroups predicting a nucleotide ligand for GPR99. By assaying known nucleotide ligands on heterologously expresd GPR99, we could not identify specifically activating substances indicating that either they are not agonists of GPR99 or that GPR99 was not expresd at the cell surface. Analysis of the chromosomal localization of all genes of the P2Y subfamily revealed that all members of subgroup "a" are encoded by less than 370 kb on chromosome 3q24, and that the genes of subgroup "b" are clustered on one hand to chromosome 11q13.5 and on the other on chromosome 3q24-25.1 clo to the subgroup "a" position. Therefore, the P2Y subfamily is a striking example for local gene amplification.
Conclusion
We identified a new orphan receptor, GPR99, with homology to the family of G protein-coupled nucleotide receptors. Phylogenetic analysis parates this family into different subgroups predicting a nucleotide ligand for GPR99.
Background
The superfamily of G protein-coupled receptors (GPRs) is one of the largest human gene families [1]. Many different approaches have been undertaken to identify new GPRs, both biochemically and by databa arches. Recently, we have identified veral new GPR genes from the databa of expresd quence tags by a complex computational strategy [2]. With the availability of the human genomic quence another source for data mining became accessible, which is especially valuable for GPR arches, since many GPR genes contain no or only a few introns. Nevertheless, the existance of pudogenes, many of which are not transcribed or lead to truncated proteins, makes it necessary to prove the expression of each putative gene found in the genome. For this publication we ud some of the new receptors with homology to the subfamily of P2Y nucleotide receptors [2] to arch for further GPRs in the human genomic databa.
Results and discussion
Identification of GPR99
Performing TBLASTN arches of the human genomic databa with the known nucleotide GPRs we identified an additional ORF, which we named GPR99. The ORF reaches from bp 140188 to 141201 of the BAC clone with the accession number AC026756.15. The putative start codon in a Kozak context is located 15 bp behind an in-frame stop codon. The last 20 bp of the ORF and the 3’ UTR are found on an EST clone derived from thyroid epithelium
电气施工员(AW827323). Sequencing the entire EST clone from the I.M.A.G.E consortium [3], which was supplied by the Resource Center of the German Human Genome Project at the Max-Planck-Institute for Molecular Genetics, confirmed the genomic quence behind the coding region for transmembrane domain six. To show mRNA expression of full-length GPR99, we amplified the entire coding region together with the upstream stop codon from a human
placenta cDNA library by PCR. Direct quencing of the PCR product was in perfect accordance with the genomic data. The quence was submitted to GenBank under accession number AF370886. mRNA expression of GPR99 was proven by northern blot analysis. A 3.0 kb mRNA was detected in kidney and, to a lower extend, in placenta (Fig. 1).
Sequence analysis
GPR99 shares 36% identical amino acids both with the P2Y1receptor (57% similarity) and with GPR91 (55% similarity) as its clost homologs (Fig. 2). "Fingerprint" analysis for GPR subtypes [4] also groups GPR99 into the P2 purinoceptor subfamily. No receptors from other species with a similar degree of relatedness were identified in the databas.
见好就收Phylogenetic analysis of all human members of the P2Y subfamily of GPRs results in three subgroups designated "a", "b", and "n" (Fig. 3A). Subgroup "n" (for n on-nucleotide receptors) contains P2Y5,P2Y7,P2Y9,and P2Y10,all of which, for homology reasons, were designated P2Y receptors. Clor analysis revealed that none of the is activated by nucleotides, but one of them, P2Y7,binds leukotriene B4 [5]. In contrast, GPR99 belongs to subgroup "b" which, in addition to the new orphan receptor GPR91, consists of five receptors with proven nucleotide agonists, hinting at a similar ligand for GPR91 and GPR99. Subgroup "a" consists of the orphan receptors GPR87 and H963, and of GPR86/P2Y13 [6], P2Y12 and KIAA0001, which bind the nucleotide ligands ADP, ADP, and UDP-gluco, respectively.
A clor analysis of residues implicated in direct nucleotide interactions confirms the existance of tw
o distinct nucleotide receptor subgroups. At the beginning of transmembrane domain ven, all receptors of subgroup "b", including GPR99, share the motif Y-X-V-T-R-P-L, which is not found in the other GPRs (Fig. 3B). The highly specific positively charged arginine residue of this motif was shown to play an important role for binding of negatively charged nucleotides [7]. In the same position the subgroup "a" members share the motif K-E-X-T-L-X-L. P2Y5,P2Y7,P2Y9,P2Y10 and other non-nucleotide receptors lack both of the
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