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.

Interleukin-8 receptor antagonists in pulmonary diseases.

Inflammatory cells are thought to be instrumental in the pathophysiology of pulmonary diseases, and control of their recruitment and activation in the lung would appear to be an attractive strategy for therapeutic intervention. Interleukin-8 and related CXC chemokines are involved in the function of neutrophils and T cells, and have been implicated in several lung diseases. Small-molecule antagonists of the interleukin-8 receptors have been identified, which may help elucidate the role of interleukin-8 and related chemokines in the pathophysiology of lung diseases.

EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes.

Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 nM, 100 nM and 1 microM. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 nM) followed by dihydro-S1P (107 nM) and sphingosylphosphorylcholine (1.6 microM). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.

AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1.

A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.

Discovery of potent and selective phenylalanine derived CCR3 antagonists. Part 1.

The discovery of a series of phenylalanine derived CCR3 antagonists is reported. Parallel, solution-phase library synthesis has been utilized to delineate the structure-activity relationship leading to the synthesis of highly potent, CCR3-selective antagonists.

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

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.

Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor.

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey.

1. Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2. The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: -log[EC(50)]s 9.09+/-0.19 and 8.84+/-0.21, R(max)s 143+/-21 and 67+/-26% 60 mM KCl, respectively (compared, for example, to -log[EC(50)] 7.90+/-0.11 and R(max) 142+/-12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (-log[EC(50)] <6.50). These findings were further contrasted by the observation that U-II was a 'coronary-selective' spasmogen in the dog (-log[EC(50)] 9.46+/-0.11, R(max) 109+/-23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (-log[EC(50)]s range from 8.96+/-0.15 to 9.92+/-0.13, R(max)s from 43+/-16 to 527+/-135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3. Thus, human U-II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U-II influences the physiological regulation of mammalian cardiovascular function.

Nonpeptide tachykinin receptor antagonists. II. Pharmacological and pharmacokinetic profile of SB-222200, a central nervous system penetrant, potent and selective NK-3 receptor antagonist.

The pharmacological and pharmacokinetic profile of SB-222200 [(S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car boxami de], a human NK-3 receptor (hNK-3R) antagonist, was determined. SB-222200 inhibited (125)I-[MePhe(7)]neurokinin B (NKB) binding to Chinese hamster ovary (CHO) cell membranes stably expressing the hNK-3 receptor (CHO-hNK-3R) with a K(i) = 4.4 nM and antagonized NKB-induced Ca(2+) mobilization in HEK 293 cells stably expressing the hNK-3 receptor (HEK 293-hNK-3R) with an IC(50) = 18.4 nM. SB-222200 was selective for hNK-3 receptors compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 250 nM). In HEK 293 cells transiently expressing murine NK-3 receptors (HEK 293-mNK-3R), SB-222200 inhibited binding of (125)I-[MePhe(7)]NKB (K(i) = 174 nM) and antagonized NKB (1 nM)-induced calcium mobilization (IC(50) = 265 nM). In mice oral administration of SB-222200 produced dose-dependent inhibition of behavioral responses induced by i.p. or intracerebral ventricular administration of the NK-3 receptor-selective agonist, senktide, with ED(50) values of approximately 5 mg/kg. SB-222200 effectively crossed the blood-brain barrier in the mouse and rat. The inhibitory effect of SB-222200 against senktide-induced behavioral responses in the mouse correlated significantly with brain, but not plasma, concentrations of the compound. Pharmacokinetic evaluation of SB-222200 in rat after oral administration (8 mg/kg) indicated sustained plasma concentrations (C(max) = about 400 ng/ml) and bioavailability of 46%. The preclinical profile of SB-222200, demonstrating high affinity, selectivity, reversibility, oral activity, and central nervous system penetration, suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors, in particular in the central nervous system.

Identification of potent, selective non-peptide CC chemokine receptor-3 antagonist that inhibits eotaxin-, eotaxin-2-, and monocyte chemotactic protein-4-induced eosinophil migration.

Eosinophils have been implicated in the pathogenesis of asthma and other allergic diseases. Several CC chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES (CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating peptides acting through CC chemokine receptor-3 (CCR3). Thus, antagonism of CCR3 could have a therapeutic role in asthma and other eosinophil-mediated diseases. A high throughput, cellular functional screen was configured using RBL-2H3 cells stably expressing CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A small molecule CCR3 antagonist was identified, SK&F 45523, and chemical optimization led to the generation of a number of highly potent, selective CCR3 antagonists including SB-297006 and SB-328437. These compounds were further characterized in vitro and demonstrated high affinity, competitive inhibition of (125)I-eotaxin and (125)I-MCP-4 binding to human eosinophils. The compounds were potent inhibitors of eotaxin- and MCP-4-induced Ca(2+) mobilization in RBL-2H3-CCR3 cells and eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis induced by three ligands that activate CCR3 with similar potencies. Selectivity was affirmed using a panel of 10 seven-transmembrane receptors. This is the first description of a non-peptide CCR3 antagonist, which should be useful in further elucidating the pathophysiological role of CCR3 in allergic inflammatory diseases.

Evidence that the proposed novel human "neurokinin-4" receptor is pharmacologically similar to the human neurokinin-3 receptor but is not of human origin.

There have been proposals that the tachykinin receptor classification should be extended to include a novel receptor, the "neurokinin-4" receptor (NK-4R), which has a close homology with the human NK-3 receptor (hNK-3R). We compared the pharmacological and molecular biological characteristics of the hNK-3R and NK-4R. Binding experiments, with (125)I-[MePhe(7)]-NKB binding to HEK 293 cell membranes transiently expressing the hNK-3R (HEK 293-hNK-3R) or NK-4R (HEK 293-NK-4R), and functional studies (Ca(2+) mobilization in the same cells) revealed a similar profile of sensitivity to tachykinin agonists and antagonists for both receptors; i.e., in binding studies with the hNK-3R, MePhe(7)-NKB > NKB > senktide >> NKA = Substance P; with the NK-4R, MePhe(7)-NKB > NKB = senktide >> Substance P = NKA; and with antagonists, SB 223412 = SR 142801 > SB 222200 >> SR 48968 >> CP 99994 for both hNK-3R and NK-4R. Thus, the pharmacology of the two receptors was nearly identical. However, attempts to isolate or identify the NK-4R gene by using various molecular biological techniques were unsuccessful. Procedures, including nested polymerase chain reaction studies, that used products with restriction endonuclease sites specific for either hNK-3R or NK-4R, failed to demonstrate the presence of NK-4R in genomic DNA from human, monkey, mouse, rat, hamster, or guinea pig, and in cDNA libraries from human lung, brain, or heart, whereas the hNK-3R was detectable in the latter libraries. In view of the failure to demonstrate the presence of the putative NK-4R it is thought to be premature to extend the current tachykinin receptor classification.

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.

Identification and molecular characterization of rat CXCR3: receptor expression and interferon-inducible protein-10 binding are increased in focal stroke.

We describe here the cloning and characterization of a rat homolog of the chemokine receptor CXCR3. The predicted amino acid sequence of rat CXCR3 contains 367 amino acid residues, sharing 96 and 87% amino acid sequence identity to the murine and human CXCR3, respectively. Among a large panel of chemokines tested, only interferon-inducible protein-10 (IP-10), interferon-gamma-induced monokine, and interferon-inducible T cell alpha-chemoattractant demonstrated specific abilities to induce an intracellular calcium mobilization response in human embryonic kidney 293 cells transfected with rat CXCR3 expression vector. (125)I-IP-10 competition binding studies to the CXCR3-transfected human embryonic kidney 293 cells demonstrated that human IP-10 and interferon-inducible T cell alpha-chemoattractant are more potent ligands than human interferon-gamma-induced monokine. Following our previous observation for the induced expression of IP-10 in focal stroke, we demonstrate here the time-dependent up-regulation of CXCR3 mRNA in the rat ischemic cortex after permanent occlusion of the middle cerebral artery. A significant increase in (125)I-IP-10-specific binding to ischemic cerebral cortical samples was obtained and paralleled the increase in CXCR3 mRNA expression. The changes in receptor expression and ligand binding correlate highly with known changes in leukocyte accumulation, and gliosis occurred after focal stroke. These data suggest that CXCR3/IP-10 may be a potential novel therapeutic target in focal stroke. In addition, the cloning of rat CXCR3 provides an important tool for the investigation of the pathophysiological role of CXCR3 in other rodent disease models.

Calmodulin-binding peptide PEP-19 modulates activation of calmodulin kinase II In situ.

PEP-19 is a 6 kDa polypeptide that is highly expressed in select populations of neurons that sometimes demonstrate resistance to degeneration. These include the granule cells of the hippocampus and the Purkinje cells of the cerebellum. Its only identified activity to date is that of binding apo-calmodulin. As a consequence, it has been demonstrated to act as an inhibitor of calmodulin-dependent neuronal nitric oxide synthase in vitro, although PEP-19 regulation of calmodulin-dependent enzymes has never been characterized in intact cells. The activation of the calmodulin-dependent enzyme calmodulin kinase II (CaM kinase II) was studied in PC12 cells that had been transfected so as to express physiological levels of PEP-19. The expression of PEP-19 yielded a stable phenotype that failed to activate CaM kinase II upon depolarization in high K(+). However, CaM kinase II could be fully activated when calcium influx was achieved with ATP. The effect of PEP-19 on CaM kinase II activation was not attributable to changes in the cellular expression of calmodulin. The cellular permeability of the transfected cells to calcium ions also appeared essentially unchanged. The results of this study demonstrated that PEP-19 can regulate CaM kinase II in situ in a manner that was dependent on the stimulus used to mobilize calcium. The selective nature of the regulation by PEP-19 suggests that its function is not to globally suppress calmodulin activity but rather change the manner in which different stimuli can access this activity.

A G protein-coupled receptor for UDP-glucose.

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

Cloning and characterization of a rabbit ortholog of human Galpha16 and mouse G(alpha)15.

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.