Publisher Summary More than 1,400 human genes have been correlated with diseases. In general, these are single-gene disorders that arise because of mutations that alter the function of a specific protein or lead to its complete absence in the disease phenotype. The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. The major model systems used include yeast, flies, worms, and mammals. There are many advantages to using these model organisms to identify signaling pathways activated or altered in a disease and also for drug discovery. The genomes are less redundant, and desired clone variants (mutants) can be generated and screened with both ease and speed. Saccharomyces cerevisiae is a model organism that has been used extensively to characterize cell cycle genes. Cell cycle studies performed in this organism have served as a guideline for understanding eukaryotic cell cycle progression. A wide variety of genomic approaches have been used to get insights into the molecular basis of tumorigenesis and predict clinical outcomes in mammalian cells. Oncogenesis occurs through the acquisition and selection of multiple somatic mutations, each contributing to the growth, survival, and spread of the cancer. Key attributes of the malignant phenotype—such as unchecked proliferation and cell survival—can often be “reversed” by the selective diminution of dominant oncogenes by chemical or genetic means.

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ETA- and ETB-selective ligands. This subtype has been identified in canine spleen membranes using ETB-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ETA-selective antagonists BQ123 and related cyclic pentapeptides. Binding of 125I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (Bmax) of 240.0 fmol/mg protein. Although the apparent affinities obtained with 125I-ET-1 and 125I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with 125I-ET-3 was approximately 35% of that obtained with 125I-ET-1, which indicates that canine spleen possesses both ETA and ETB receptors in the ratio 65:35. Competition binding experiments using 125I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for 125I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ETA-selective antagonist, displaced 125I-ET-3 binding from canine spleen with an IC50 value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)+ RNA indicated that the ETB receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ETB receptor present in this tissue is similar to that of the cloned human ETB receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ETB receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ETB receptors displayed different molecular weight bands and different pharmacology. In addition, the ETB receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ETB receptor in different tissues as well as different species including human.

In a human neutrophil cDNA library, an orphan G-protein-coupled receptor, HNFAG09, with 37% nucleotide identity to the C5a receptor (C5a-R, CD88) was identified. A novel feature of this gene, unlike C5a-R and other G-protein-coupled receptors, is the presence of an extraordinarily large predicted extracellular loop comprised of in excess of 160 amino acid residues between transmembrane domains 4 and 5. Northern blot analysis revealed that expression of mRNA for this receptor in human tissues, while similar, was distinct from C5a-R expression. Although there were differences in expression, transcripts for both receptors were detected in tissues throughout the body and the central nervous system. Mammalian cells stably expressing HNFAG09 specifically bound 125I-C3a and responded to a C3a carboxyl-terminal analogue synthetic peptide and to human C3a but not to rC5a with a robust calcium mobilization response. HNFAG09 encodes the human anaphylatoxin C3a receptor.

Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine2A (5-HT2A) or 5-HT2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT2C receptor (pEC50 = 7.80 +/- 0.06) compared with the 5-HT2A receptor (pEC50 = 7.30 +/- 0.08). Activation of the 5-HT2A receptor caused a transient fourfold increase in intracellular Ca2+ concentration. Whole-cell recordings of cells clamped at -50 mV demonstrated a small inward current (2 pA) in response to 10 microM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT2C receptor, whereas activity at the 5-HT2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.

The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G-protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co-injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co-injection of a pertussis toxin sensitive complementary human factor which could be supplied by co-injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.

High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.

Membranes prepared from Xenopus liver displayed high density of high affinity endothelin (ET) binding sites. These sites have the same affinity for [125I] ET-1 and [125I] ET-3. Scatchard analysis of the specific binding from saturation binding experiments revealed an apparent dissociation constant (Kd) of 93.1 and 70.9 pM and maximum binding (Bmax) of 602 and 651 fmol/mg protein for [125I] ET-1 and [125I] ET-3, respectively. Competition binding experiments using [125I] ET-1 and unlabelled ET-1, ET-3, S6c, and BQ123 indicated that ET-1 and ET-3 were the most potent in displacing [125I] ET-1 binding from these membranes (IC50 1 and 0.3 nM, respectively), whereas S6c BQ123, selective for ETB and ETA receptors, respectively, did not have any inhibitory effect up to 1 microM. These data clearly indicate that the ET receptors present in Xenopus liver membranes belong to a new subtype of ET receptor. Because it resembled mammalian ETB receptors in its affinities for ET-1 and ET-3, we propose that this receptor be called the ETBX receptor.

Endothelin (ET) receptors display subtype heterogeneity and so far three subtypes of ET receptors, namely ET(A), ET(B), and ET(C), have been identified, cloned, sequenced, and characterized. Based on the binding profile of ET and related peptides, a novel ET receptor (ET(AX)) was identified in the follicular membranes of Xenopus laevis oocytes (Kumar, C. S., Nuthulaganti, P., Pullen, M., and Nambi, P. (1993). Mol. Pharmacol. 44, 153-157). Here we report the cloning and characterization of this ET(AX) subtype from X. Laevis heart. A cDNA was isolated that encodes a protein of 415 amino acids that shares 74, 60, and 51% identities with human ET(A), human ET(B), and Xenopus ET(C) receptors, respectively. Competition binding studies of the cloned receptor expressed in COS cells using ET-related peptides suggested that this receptor is pharmacologically identical to that expressed in Xenopus oocyte follicular, heart, and lung membranes. Phosphoinositide turnover and oocyte electrophysiological studies indicated that the cloned receptor is functionally coupled to a second messenger system.

A Xenopus laevis heart cDNA library was screened using the human angiotensin type 1 (AT1) receptor cDNA coding sequence as a hybridization probe. A cDNA was isolated that encodes a protein of 363 amino acids that shares 63% sequence identity with the human AT1 receptor. Radioligand binding studies with the cloned receptor expressed in COS cells indicated that it is an angiotensin II receptor that possesses pharmacological properties distinct from those of the two known mammalian receptor subtypes, AT1 and AT2. Electrophysiological studies with the recombinant receptor expressed in X. laevis oocytes revealed that the amphibian receptor, like the mammalian AT1 receptor, can functionally couple to a second messenger system, leading to the mobilization of intracellular stores of calcium. However, nonpeptide antagonists selective for the mammalian AT1 and AT2 receptors do not block angiotensin II-stimulated functional responses in injected oocytes, which confirms that the amphibian receptor is a pharmacologically unique angiotensin II receptor. Nevertheless, based on conservation of structural features and motifs and similarity in coupling mechanisms, we speculate that the cloned Xenopus receptor is the amphibian counterpart of the mammalian AT1 receptor, having acquired its unique pharmacology as a consequence of evolutionary divergence.

Steroid hormones have been shown to modulate a number of physiological processes in addition to their potent antiinflammatory effects. Endothelin (ET) is a newly discovered vasoconstrictor that is synthesized and released by endothelial cells and acts on adjacent vascular smooth muscle cells by interacting with specific cell surface receptors. Proinflammatory agents such as thrombin and transforming growth factor beta have been shown to up-regulate ET gene expression in vascular endothelial cells. We wondered whether the anti-inflammatory steroids might have any regulatory effect on the ET receptors present in the vascular smooth muscle cells. Rat vascular smooth muscle cells (A-10 cell line, ATCC.CRL 1476) were used as a model system to study the effects of glucocorticoids on ET receptor expression and function. These cells display high density and high affinity ET receptors that belong to the ETA subtype. Pretreatment of these cells with dexamethasone reduced the number of ET receptors by 50-60% without changing the affinity. Of the steroids tested, dexamethasone was most effective followed by prednisolone and hydrocortisone. Aldosterone, a mineralocorticoid, was 5000-fold less potent than dexamethasone. This effect of dexamethasone was dependent on the time of pretreatment and concentration of the steroid used. This down-regulation of ET receptors was also accompanied by an attenuated response to ET-1 in dexamethasone-pretreated cells. The inhibitory effect of dexamethasone was selective for ET receptors because the vasopressin-mediated response was unaffected. In addition, dexamethasone pretreatment of these cells resulted in 50-60% reduction in the steady-state level of ETA receptor mRNA as revealed by Northern analysis. These results suggest that glucocorticoid pretreatment of smooth muscle cells resulted in the down-regulation of the ET(A) receptor at the mRNA level.