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.     

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.     

Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin

Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.     

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 and expression of a novel angiotensin II receptor subtype.

Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.     

2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists with good brain exposure and in vivo efficacy for plasma LH level in male rats

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor plays an essential role to modulate sex-hormones including GnRH. Thus, antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis. We recently reported 2-acylamino-4,6-diphenylpyridines as the first small molecule GPR54 antagonists with high potency. However, the representative compound 1 showed low brain exposure, where GPR54 acts as a modulator of gonadotropins by binding with its endogenous ligand, metastin. In order to discover compounds that have not only potent GPR54 antagonistic activity but also good brain permeability, we focused on converting the primary amine on the side chain to a secondary or tertiary amine, and finally we identified 15a containing a piperazine group. This compound exhibited high affinity to human and rat GPR54, apparent antagonistic activity, and high brain exposure. In addition, intravenous administration of 15a to castrated male rat suppressed plasma LH level, which indicates the possibility of a small molecule GPR54 antagonist as a novel drug for sex-hormone dependent diseases.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

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.     

Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions.

A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD. The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites. Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed. Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

GPR18 Agonist Resolvin D2 Reduces Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage by Multiple Protective Mechanisms

Early brain injury (EBI) is the early phase of secondary complications arising from subarachnoid hemorrhage (SAH). G protein-coupled receptor 18 (GPR18) can exert neuroprotective effects during ischemia. In this study, we investigated the roles of GPR18 in different brain regions during EBI using a GPR18 agonist, resolvin D2 (RvD2). Location and dynamics of GPR18 expression were assessed by immunohistochemistry and western blotting in a rat model of SAH based on endovascular perforation. RvD2 was given intranasally at 1 h after SAH, and SAH grade, brain water content and behavior were assayed before sacrifice. TUNEL and dihydroethidium staining of the cortex were performed at 24 h after SAH. Selected brain regions were also examined for pathway related proteins using immunofluorescence and Western blotting. We found that GPR18 was expressed in meninges, hypothalamus, cortex and white matter before EBI. After SAH, GPR18 expression was increased in meninges and hypothalamus but decreased in cortex and white matter. RvD2 improved neurological scores and brain edema after SAH. RvD2 attenuated mast cell degranulation and reduced expression of chymase and tryptase expression in the meninges. In the hypothalamus, RvD2 attenuated inflammation, increased expression of proopiomelanocortin and interleukin-10, as well as decreased expression of nerve peptide Y and tumor necrosis factor-α. In cortex, RvD2 alleviated oxidative stress and apoptosis, and protected the blood–brain barrier. RvD2 also ameliorated white matter injury by elevating myelin basic protein and suppressing amyloid precursor protein. Our results suggest that GPR18 may help protect multiple brain regions during EBI, particularly in the cortex and hypothalamus. Upregulating GPR18 by RvD2 may improve neurological functions in different brain regions via multiple mechanisms.