Dennis K. Lee

Characterization of Apelin, the Ligand for the APJ Receptor

Abstract: The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein‐coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25‐26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full‐length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

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.

Identification and cloning of three novel human G protein-coupled receptor genes GPR52, ΨGPR53 and GPR55: GPR55 is extensively expressed in human brain

The G protein-coupled receptor (GPCR) family share a structural motif of seven transmembrane segments with large numbers of conserved residues in those regions. Here, we report the identification and cloning of two novel human intronless GPCR genes, GPR52, GPR55 and a pseudogene PsiGPR53. GPR55 was identified from the expressed sequence tags (EST) database whereas GPR52 and pseudogene PsiGPR53 originated from the high throughput genome (HTG) database. A partial cDNA clone obtained from the IMAGE Consortium of GPR55 was used to screen a human genomic library to acquire the full length gene. GPR52 and PsiGPR53 were amplified from human genomic DNA using primers based on the HTG sequences. GPR55 and GPR52 encode receptors of 319 and 361 amino acids, respectively. GPR55 gene was mapped to chromosome 2q37, using fluorescence in situ hybridization (FISH), and its mRNA transcripts have been detected in the caudate nucleus and putamen, but not in five other brain regions. Human receptors showing the highest amino acid identity to GPR55 include P2Y5 (29%), GPR23 (30%), GPR35 (27%) and CCR4 (23%). GPR52 gene localized to chromosome 1q24 shares the highest identity with GPR21 (71%), histamine H2 (27%) and 5-HT4 (26%) human receptors. PsiGPR53 is a pseudogene mapped to chromosome 6p21 that demonstrates the highest similarity to the MRG (35%), MAS (28%) and C5a (24%) human receptor genes.

Discovery and mapping of ten novel G protein-coupled receptor genes

We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed in the human prostate and rat stomach and fetal tissues. GPR101 revealed mRNA transcripts in caudate putamen and hypothalamus. GPR103 mRNA signals were detected in the cortex, pituitary, thalamus, hypothalamus, basal forebrain, midbrain and pons.

Two related G protein-coupled receptors: The distribution of GPR7 in rat brain and the absence of GPR8 in rodents

GPR7 and GPR8, orphan G protein-coupled receptor (GPCR) genes, expressed in the brain and periphery share highest sequence identity to each other and significant similarity with opioid and somatostatin receptors. To further our knowledge of GPR7s physiological function, we performed in situ hybridization analyses of rat brain to reveal specific patterns of expression in the brain. GPR7 mRNA was found to be discretely localized in areas of the amygdala, hippocampus, hypothalamus and cortex. We previously reported that GPR7 was highly conserved in both human and rodent orthologs while GPR8 was not found in the rodent [9]. We speculated that GPR8 originated after the divergence of the human and rodent. Using primers designed from human GPR8, we isolated lemur GPR8 and subsequently aligned human, monkey, and lemur GPR8 orthologs to design primers recognizing highly conserved regions of GPR8. Using these primers, orthologs of GPR7 and GPR8 were isolated by the PCR from rabbit, tree shrew, and flying lemur, as well as GPR7 in the rat. Subsequent analysis of the clones obtained demonstrated that both GPR7 and GPR8 sequences were highly conserved amongst the species studied, but a rodent GPR8 was not isolated. The absence of a GPR8 gene in the rodent suggests that GPR8 originated from gene duplication of GPR7 after the rodent line diverged from the rabbit, tree shrew, flying lemur, lemur, monkey and human lines. In addition, the taxonomic distribution of GPR8 is consistent with molecular studies grouping rabbits with primates, tree shrews and flying lemurs rather than with rodents.

Orphan G protein-coupled receptors in the CNS

The majority of genes encoding G protein-coupled receptors were isolated by methods based on sequence similarities found throughout this family. Experimental techniques have exploited these similarities (including low-stringency hybridization, polymerase chain reaction and electronic database searching) to identify genes encoding many pharmacologically recognized receptors and their subtypes. Homology-based searches have revealed receptors for which the endogenous ligands were unknown and these were named orphan receptors. Many orphan receptors are expressed in the brain, suggesting the existence of unidentified neurotransmitters. Methods used to identify ligands for these orphan receptors resulted in the identification of novel ligands and succeeded in pairing previously identified ligands with their receptors. Similar successful strategies are required to characterize the physiological and pathological importance of the remaining orphan receptors to facilitate the discovery of novel drugs for these systems.

Identification of four novel human G protein-coupled receptors expressed in the brain.

We report the discovery and tissue distributions of four novel human genes, GPR61, GPR62, GPR63 and GPR77, all of which encode G protein-coupled receptors (GPCRs). GPR61 was discovered in a search of the patent literature which retrieved a rabbit DNA sequence partially encoding a novel GPCR. This sequence was used to obtain a full-length human cDNA encoding GPR61, a receptor of 417 amino acid length. A search of the GenBank genomic sequence databases revealed three previously unrecognized intronless genes encoding the orphan GPCrs (oGPCRs) GPR62, GPR63 and GPR77, with respective amino acid lengths of 368, 419 and 337. Sequence analysis revealed that GPR61 and GPR62, and a published orphan receptor p47MNR, shared the highest level of identities to each other, ranging from 36 to 45% in the transmembrane (TM) domains. Together, these three oGPCRs appear to comprise a novel subfamily of GPCRs, most closely related to the serotonin 5-HT(6) receptor. Sequence analysis of GPR63 and GPR77 revealed highest sequence identities in the TM regions with the oGPCR PSP24 (58%) and the anaphylatoxin C5a receptor (49%) respectively. Tissue distribution analyses detected the expression of all four novel genes in the human brain. GPR61 mRNA expression was detected in the caudate, putamen and thalamus of human brain, with a more widespread expression pattern in rat brain, with mRNA signals in areas of the cortex, hippocampus, thalamus, hypothalamus and midbrain. GPR62 mRNA expression was detected in the basal forebrain, frontal cortex, caudate, putamen, thalamus and hippocampus. GPR63 mRNA expression was detected in the frontal cortex, with lower levels in the thalamus, caudate, hypothalamus and midbrain. Analysis of GPR77 mRNA expression revealed signals in the frontal cortex, hippocampus and hypothalamus with high transcript levels in the liver.

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.

The fate of the internalized apelin receptor is determined by different isoforms of apelin mediating differential interaction with β-arrestin

Internalization of the apelin receptor by apelin-13 is characterized by dissociation from beta-arrestins and rapid recycling to the cell surface. Paradoxically, the apelin receptor internalized by apelin-36 was sequestered intracellularly. The specific pathways involved in apelin receptor trafficking were resolved using beta-arrestin1 and constitutively active and dominant negative Rab proteins following activation by apelin-13 or apelin-36. beta-Arrestin1 dissociated from the apelin-13-internalized receptor while the apelin-36-internalized receptor was trafficked with beta-arrestin1 to intracellular compartments. The apelin-13-internalized receptor was rapidly recycled to the cell surface through a Rab4-dependent mechanism while Rab7 targeted the receptor to lysosomes. The internalized receptor co-expressed with dominant negative Rab4 were trafficked to lysosomes. These observations revealed a novel ligand-dependent targeting of the apelin receptor to beta-arrestin-associated and -dissociated trafficking pathways and a role for different Rab proteins to direct these pathways.

Novel G-protein-coupled receptor genes expressed in the brain: continued discovery of important therapeutic targets

The rhodopsin family of G-protein-coupled receptors (GPCRs) is the largest known group of cell-surface mediators of signal transduction. The vast majority of these receptors were discovered by methods based upon shared sequence homologies found throughout this family. While such efforts identified a multitude of receptor subtypes for previously known ligands, numerous receptors have been discovered for which endogenous ligands were unknown. These receptors are commonly referred to as orphan receptors. One of the most important tasks of modern pharmacology lies in elucidating the functions of these receptors. Of particular interest are receptors with recognised expression in the central nervous system, given that many psychiatric and neurodegenerative disorders are mediated by unknown mechanisms. Hence, this collection of putative neurotransmitter and neuromodulator signal mediators represents a substantial and untapped resource for novel drug discovery. Recently, various methodologies have accelerated the discovery of novel ligands for these orphan receptors, identifying the basic components required for further physiological ligand/receptor system characterisation. Equipped with proven ligand identification strategies, the characterisation of all orphan GPCRs and the exploitation of their exciting potential as targets for the discovery of novel drugs is anticipated.