Cloning, expression, and pharmacological characterization of a novel human histamine receptor.

Using a genomics-based reverse pharmacological approach for screening orphan G-protein coupled receptors, we have identified and cloned a novel high-affinity histamine receptor. This receptor, termed AXOR35, is most closely related to the H3 histamine receptor, sharing 37% protein sequence identity. A multiple responsive element/cyclic AMP-responsive element-luciferase reporter assay was used to identify histamine as a ligand for AXOR35. When transfected into human embryonic kidney 293 cells, the AXOR35 receptor showed a strong, dose-dependent calcium mobilization response to histamine and H3 receptor agonists including imetit and immepip. Radioligand binding confirmed that the AXOR35 receptor was a high-affinity histamine receptor. The pharmacology of the AXOR35 receptor was found to closely resemble that of the H3 receptor; the major difference was that (R)-alpha-methylhistamine was a low potency agonist of the AXOR35 receptor. Thioperamide is an antagonist at AXOR 35. Expression of AXOR35 mRNA in human tissues is highest in peripheral blood mononuclear cells and in tissues likely to contain high concentrations of blood cells, such as bone marrow and lung. In situ hybridization analysis of a wide survey of mouse tissues showed that mouse AXOR35 mRNA is selectively expressed in hippocampus. The identification and localization of this new histamine receptor will expand our understanding of the physiological and pathological roles of histamine and may provide additional opportunities for pharmacological modification of these actions.

Identification of an EDG7 variant, HOFNH30, a G-protein-coupled receptor for lysophosphatidic acid.

We have identified a cDNA, designated HOFNH30, which encodes a 354 amino acid G-protein-coupled receptor (GPCR). This receptor has 96% amino acid identity to the Jurkat-T cell-derived EDG7 and could be a splice variant. RT-PCR analysis demonstrated that HOFNH30 mRNA is expressed in placenta whereas EDG7 mRNA shows highest expression in prostate. The HOFNH30 gene is localized to human chromosome 1p22. 3-1p31.1. When HOFNH30 was expressed in RBL-2H3 cells, LPA and phosphatidic acid (PA) induced a calcium mobilization response with EC(50) values of 13 nM and 3 microM, respectively. LPA also induced phosphorylation of mitogen-activated protein kinase (p42(MAPK) and p44(MAPK)) in HOFNH30-transfected but not vector-transfected RBL-2H3 cells. In the present study, we have identified a novel variant from the EDG receptor family, a GPCR for which LPA is a high-affinity endogenous ligand.

Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor.

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay.

We have established a rapid, sensitive, high-throughput assay that requires one assay condition to detect agonist effects from Gi-, Gs-, and Gq-coupled receptors. We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP response element controlling the transcription of the luciferase gene. An adrenergic agonist, para-aminoclonidine, inhibited forskolin-stimulated luciferase expression when cells were cotransfected with the Gi-coupled alpha(2)-C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonstrate that gastrin-releasing peptide, which activates a Gq-coupled GRP receptor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of protein kinase C, can mediate increases in luciferase expression in the presence of forskolin but not in its absence. The effect at Gi-coupled receptor activation correlates with the phosphorylation of the CRE binding protein (CREB); however, the mechanisms mediating the responses to Gq- and Gs-coupled receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the potential to associate orphan G-protein-coupled receptors with their corresponding ligands.

Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor.

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1.

The underlying causes of obesity are poorly understood but probably involve complex interactions between many neurotransmitter and neuropeptide systems involved in the regulation of food intake and energy balance. Three pieces of evidence indicate that the neuropeptide melanin-concentrating hormone (MCH) is an important component of this system. First, MCH stimulates feeding when injected directly into rat brains; second, the messenger RNA for the MCH precursor is upregulated in the hypothalamus of genetically obese mice and in fasted animals; and third, mice lacking MCH eat less and are lean. MCH antagonists might, therefore, provide a treatment for obesity. However, the development of such molecules has been hampered because the identity of the MCH receptor has been unknown until now. Here we show that the 353-amino-acid human orphan G-protein-coupled receptor SLC-1 expressed in HEK293 cells binds MCH with sub-nanomolar affinity, and is stimulated by MCH to mobilize intracellular Ca2+ and reduce forskolin-elevated cyclic AMP levels. We also show that SLC-1 messenger RNA and protein is expressed in the ventromedial and dorsomedial nuclei of the hypothalamus, consistent with a role for SLC-1 in mediating the effects of MCH on feeding.