Brian F. O'Dowd

Characterization of the Human Cysteinyl Leukotriene 2 Receptor

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC4, LTD4, and LTE4, are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT1 receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT2, a 346-amino acid protein with 38% amino acid identity to the CysLT1 receptor. The recombinant human CysLT2 receptor was expressed in Xenopusoocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC4, LTD4, or LTE4. Analyses of radiolabeled LTD4 binding to the recombinant CysLT2 receptor demonstrated high affinity binding and a rank order of potency for competition of LTC4 = LTD4 ≫ LTE4. In contrast to the dual CysLT1/CysLT2 antagonist, BAY u9773, the CysLT1 receptor-selective antagonists MK-571, montelukast (SingulairTM), zafirlukast (AccolateTM), and pranlukast (OnonTM) exhibited low potency in competition for LTD4 binding and as antagonists of CysLT2receptor signaling. CysLT2 receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11

We report the cloning of a gene, intronless in its coding region, which we have named APJ. This gene was cloned using the polymerase chain reaction (PCR), with a set of primers designed on the basis of the conservation that members of G protein-coupled receptors (GPCR) have in their transmembrane (TM) regions. The putative receptor protein, APJ, shares closest identity to the angiotensin receptor (AT1) ranging from 40 to 50% in the hydrophobic TM regions of these receptors. The transcripts for this gene were detected in many regions of the brain. PCR analysis of somatic cell lines found APJ-related sequences to be only present on chromosome 11, and high-resolution mapping by fluorescence in situ hybridization (FISH) sublocalized APJ on band q12.

G-protein-coupled receptor oligomerization and its potential for drug discovery.

G-protein-coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. Virtually all therapeutics that are directed towards GPCRs have been designed using assays that presume that these receptors are monomeric. The recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. However, this important aspect of GPCR biology remains largely unincorporated into schemes to search for new therapeutics. This review provides a synopsis of the current thinking surrounding GPCR homo-oligomerization and hetero-oligomerization and shows how new models point towards unexplored avenues in the development of new therapies.

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.

D1–D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum

We demonstrate a heteromeric D1–D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1–D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1–D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase IIα in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1–D2 dopamine receptor complexes may contribute to synaptic plasticity.

Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the Gq pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.

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.

Agonist-independent Nuclear Localization of the Apelin, Angiotensin AT1, and Bradykinin B2 Receptors

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT1 and bradykinin B2 receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT1 and B2 receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT1, and B2 receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT1, and B2 receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.