Cloning and Characterization of Two Human G Protein-Coupled Receptor Genes (GPR38 and GPR39) Related to the Growth Hormone Secretagogue and Neurotensin Receptors

The recent cloning of a growth hormone secretagogue receptor (GHS-R) from human pituitary gland and brain identified a third G protein-coupled receptor (GPC-R) involved in the control of growth hormone release. The nucleotide sequence of the GHS-R is most closely related to the neurotensin receptor-1 (NT-R1) (35% overall protein identity). Two human GPC-Rs related to both the type 1a GHS-R and NT-Rs were cloned and characterized. Hybridization at low posthybridizational stringency with restriction enzyme-digested human genomic DNA resulted in the identification of a genomic clone encoding a first GHS-R/NT-R family member (GPR38). A cDNA clone was identified encoding a second GHS-R-related gene (GPR39). GPR38 and GPR39 share significant amino acid sequence identity with the GHS-R and NT-Rs 1 and 2. An acidic residue (E124) in TM-3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, was conserved in GPR38 and GPR39. GPR38 is encoded by a single gene expressed in thyroid gland, stomach, and bone marrow. GPR39 is encoded by a highly conserved single-copy gene, expressed in brain and other peripheral tissues. Fluorescencein situhybridization localized the genes for GPR38 and GPR39 to separate chromosomes, distinct from the gene encoding the GHS-R and NT-R type 1. The ligand-binding and functional properties of GPR38 and GPR39 remain to be determined.     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

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.     

A Cluster of Four Novel Human G Protein-Coupled Receptor Genes Occurring in Close Proximity to CD22 Gene on Chromosome 19q13.1

In our search for novel human galanin receptor (GALR) subtypes, human genomic DNA was PCR amplified using sets of degenerate primers based on conserved sequences in human and rat GALR. The sequence of one of the subcloned PCR products revealed homology to a sequence in the 3′ region of the human CD22 gene following a BLAST search of GenBank's database. A search for open reading frames (ORF) in the non-coding CD22 sequence resulted in identification of two novel putative intronless genes, GPR40 and GPR41. The recent submission of sequence overlapping the downstream CD22 sequence revealed a possible polymorphic insert containing a third intronless gene, GPR42, sharing 98% amino acid identity with GPR41, followed by a fourth intronless gene, GPR43. Thus, the GPR40, GPR41, GPR42, and GPR43 genes, respectively, occur downstream from CD22, a gene previously localized on chromosome 19q13.1. The four putative novel human genes encode new members of the GPCR family and share little homology with GALR.     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

The rise and fall of the chemoattractant receptor GPR33

Chemokine and chemoattractant receptors are members of the large superfamily of G protein-coupled receptors (GPCR), which control leukocyte chemotaxis. In addition to their physiological role, several chemokine and chemoattractant receptors, such as CCR5 and Duffy, have been directly associated with pathogen entry. GPR33 is an orphan chemoattractant GPCR that was previously identified as a pseudogene in humans. GPR33 evolved in mammals about 125-190 million years ago. The cloning and analysis of more than 120 mammalian GPR33 orthologs from 16 of 18 eutherian orders revealed an inactivation of this chemoattractant GPCR not only in humans, but also in several great ape and rodent species. Intriguingly, in all ape and some rodent species where the inactivation occurred, samples harbored both pseudogene and intact gene variants. The analysis of over 1200 human individuals representing all major linguistic groups revealed that the intact allele of GPR33 is still present in the human population. Estimates of the age of the human alleles suggest inactivation in the past 1 million years. Similarly, analysis of more than 120 wild-caught gray rats (Rattus norvegicus), revealed that inactivation of gpr33 is worldwide fixed and occurred in less than 0.7 million years ago. The coincidental inactivation and its fixation in several species of distantly related mammalian orders suggest a selective pressure on this chemoattractant receptor gene.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

A Novel Human Gene Encoding a G-Protein-Coupled Receptor (GPR15) Is Located on Chromosome 3

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have namedGPR15.A comparison of the amino acid sequence of the receptor encoded byGPR15with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1.GPR15was mapped to human chromosome 3q11.2–q13.1.     

Discovery of a receptor related to the galanin receptors

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.     

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3–24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22–q31.1, and GPR23 to chromosome X, region q13–q21.1.© 1997 Elsevier Science B.V. All rights reserved.     

A G protein-coupled receptor encoded by rhesus rhadinovirus is similar to ORF74 of Kaposi's sarcoma-associated herpesvirus

Rhesus rhadinovirus (RRV) is a gamma-2 herpesvirus and is the rhesus macaque homologue of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. DNA sequence analysis of RRV indicates that it shares numerous open reading frames (ORFs) with HHV-8, including one (ORF74) encoding a seven-transmembrane-spanning G protein-coupled receptor (GPCR) with similarity to cellular chemokine receptors. Examination of the predicted amino acid sequence of RRV ORF74 reveals that it encodes a seven-transmembrane-spanning GPCR sharing 40.8% amino acid sequence identity with HHV-8 ORF74 and 24.1% amino acid sequence identity with rhesus macaque CXCR2. In addition, immunofluorescence studies indicate that an epitope-tagged version of RRV ORF74 is expressed on the surfaces of transfected cells, suggesting that this protein is in fact a membrane receptor. In in vitro cell culture assays, RRV ORF74 possesses transforming potential, as NIH 3T3 clones stably expressing the receptor demonstrate an increased ability to grow in soft agarose and to induce tumor formation in nude mice. Further analysis of RRV ORF74 indicates that expression of the receptor in NIH 3T3 cells causes an increased secretion of vascular endothelial growth factor and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway. The results of these studies suggest that RRV ORF74 encodes a GPCR with properties similar to those of its homologue in HHV-8 and that this gene may play a role in RRV-associated pathogenesis.     

Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.     

Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver

By homology search of expressed sequence tags (EST) in GenBank a novel member of the CC chemokine family was identified. The full-length sequence of this liver-specific CC chemokine (LCC-1) predicted a mature protein of 97 amino acids with 31-48% identity to other CC chemokines. There was a characteristic amino acid C-term extension when aligned with other chemokines. Northern blot analysis from a panel of human tissues revealed that LCC-1 mRNA expression is restricted to adult and fetal liver. Different polyadenylation results in two mRNA species of 1.5 kb and 0.5 kb in size. LCC-1 is constitutively expressed in human HepG2 hepatoma cells and is induced by hypoxic exposure. The promoter region of the LCC-1 gene contains potential HIF-1 binding sites. The EST for LCC-1 has been previously mapped to the CC chemokine cluster on human chromosome 17q11.2. The organization of the LCC-1 gene (scya16) into three exons interrupted by two introns is identical to that found for other members of the CC chemokine family.     

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.