Research reportIdentification and cloning of three novel human G protein-coupled receptor genes GPR52, ΨGPR53 and GPR55: GPR55 is extensively expressed in human brain1
Introduction
The GPCR family plays an important role in signal transduction from the external environment to the inside of the cell thus regulating intracellular signaling and gene expression. This receptor family responds to a broad spectrum of extracellular ligands, including nucleotides, peptides, biogenic amines, glycoprotein hormones or light, and amplifies the signal inside the cell by interacting with heterotrimeric GTP-binding proteins 12, 13. The GPCRs, integral cell membrane proteins, share the characteristic structural motif of seven transmembrane (TM) domains that comprise the majority of conserved amino acid motifs in the family. The motif conservation among the GPCR family members has been exploited by performing low-stringency hybridization and degenerate PCR to identify and clone genes encoding novel GPCRs. The recently introduced GenBank High Throughput Genome (HTG) database contains sequences from genome sequencing projects of Homo sapiens, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Mus musculus. The sequences deposited into the GenBank HTG division comprise of sequence fragments from single genomic contigs. These sequences can contain unorganized fragments, gaps between fragments and sequencing errors. This division of the database provides an excellent resource for the identification and genomic characterization of novel genes that have low expression, are expressed transiently in discrete tissue sources or are not expressed at all in contrast to genes represented in the expressed sequence tags (EST) database. The homology searches of these databases with sequences of known GPCRs `cyberprobes' [3]is an excellent addition to currently used methods including low-stringency library screening or degenerate PCR.
By performing homology searches, using BLAST (basic local alignment search tool) with amino acid sequences of known GPCRs, of publicly available databases such as HTG or EST databases we were able to identify `in silico' [3]and clone two novel human genes GPR55, GPR52 and a pseudogene ΨGPR53 belonging to the GPCR family.
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Database searching
We queried the EST and HTG databases maintained by the National Center for Biotechnology Information (NCBI), with various amino acid sequences encoding rhodopsin family GPCRs using the BLAST algorithm [1]. Sequences that were returned having statistically significant scores (E values<0.05) were searched manually to determine whether highly conserved amino acid sequence motifs characteristic for GPCRs were present in the translated sequences. The predicted amino acid sequences were then used to
Cloning GPR55
As part of our continuing search for novel genes encoding GPCRs, we queried the EST database with known GPCR sequences. One EST cDNA clone (clone ID: 248165) appeared to partially encode a novel GPCR gene. Originally isolated from a human fetal liver spleen cDNA library, this EST, ∼350 bp in pT7T3 vector, was obtained from the I.M.A.G.E. Consortium. Further sequencing of this EST revealed a DNA fragment partially encoding a novel GPCR TM7 domain and continuing towards the 3′-untranslated
Discussion
Hydrophobic analysis of the deduced amino acid sequences encoded by GPR55 and GPR52 genes revealed seven hydrophobic regions corresponding to the seven putative TM regions, a conserved structural motif characteristic for the GPCR family members [11]. ΨGPR53 encodes a receptor truncated at TM4. Conserved features present in the receptors include asparagine in TM1, aspartate in TM2 and conserved prolines in TM4 to TM7 regions [11]. Other characteristic hallmarks of GPCRs include conserved first
Acknowledgements
This research was supported by grants from the National Institute on Drug Abuse, the Medical Research Council of Canada, P.M.A.C. grant from Merck Frosst (Canada) and the Smokeless Tobacco Research Council to B.F.O. and S.R.G. M.S. was supported with a studentship from the Medical Research Council of Canada.
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Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF096784-AF096786, AF100789.