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WO1991015583A1 - Famille de proteines apparentee a une proteine precoce immediate exprimee par des cellules endotheliales humaines durant la differenciation - Google Patents

Famille de proteines apparentee a une proteine precoce immediate exprimee par des cellules endotheliales humaines durant la differenciation Download PDF

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WO1991015583A1
WO1991015583A1 PCT/US1991/002344 US9102344W WO9115583A1 WO 1991015583 A1 WO1991015583 A1 WO 1991015583A1 US 9102344 W US9102344 W US 9102344W WO 9115583 A1 WO9115583 A1 WO 9115583A1
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protein
edg
endothelial cells
receptor
cells
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Timothy Tun Hla
Thomas Maciag
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American National Red Cross
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American National Red Cross
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • Endothelium is composed of a monolayer of quiescent cells, endothelial cells.
  • Endothelial cells which form the inner lining of blood vessels participate in a multiplicity of physiological functions, including the formation of a. selective barrier for the translocation of blood constituents and macromolecules to underlying tissues and the maintenance of a non-thrombogenic interface between blood and tissue.
  • Endothelial cells are also an important component in the development of new capillaries and blood vessels.
  • Blood vessel development, which is called angiogenesis occurs during developmental periods, such as during development of the vascular system, and as part of the pathophysiology of a variety of disease .
  • Angiogenesis which involves the organized migration, proliferation, and differentiation of the endothelial cells, is initiated by the endothelial cell in response to angiogenic stimuli and can be separated into three distinct events: cell migration, cell proliferation and cell differentiation, whereby the cells organize into a tubular structure. These events are mediated in vitro. and most likely in vivo, by mitogenic polypeptides.
  • the migration of endothelial cells is induced by factors, including the heparin binding growth factors and angiotropin.
  • HBGFs heparin binding growth factors
  • HBGFs heparin binding growth factors
  • polypeptides including interleukin-1 (hereinafter IL-1) , tumor necrosis factor (hereinafter TNF) , gamma-interferon, transforming growth factor alpha and beta (hereinafter TGF- ⁇ and TGF- ⁇ , respectively) and phorbol mistric acetate (hereinafter PMA) .
  • IL-1 interleukin-1
  • TNF tumor necrosis factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factor alpha and beta
  • PMA phorbol mistric acetate
  • ECM extracellular matrix
  • ECM extracellular matrix
  • endothelial cells are cultured in vitro on collagen gels in the presence of PMA organized networks of tubular structures form, and, if the cells are cultured in ECM conditioned medium the formation of tubular structures is accelerated.
  • ECM components for mediation of endothelial cell differentiation is evidenced by the observations that antibodies that have been prepared against fibronectin and laminin inhibit formation of the differentiated phenotype, while proteolytic modification of fibronectin by plasmin leads to rapid modification of the endothelial cell phenotypic changes that are observed in vitro.
  • competitive inhibitors of the laminin and fibronectin receptor binding domains ' also inhibit the ability of endothelial cells to complete the non-terminal differentiation program.
  • the polypeptide cytokines and PMA inhibit the HBGF-1-induced proliferation of endothelial cells and induce differentiation thereof. These factors induce a reversible phenotypic transition from a non-polar cobblestone monolayer into a polar elongated, fibroblast-like phenotype.
  • the inhibition of HBGF-1-induced proliferation is mediated, at least in part, via down regulation of the HBGF-1 receptor.
  • PMA activates protein kinase C, which a family of phospholipid- and calcium-activated protein kinases.
  • This activation results in the transcription of an array of proto-oncogene transcription factors, including c- os, c-myc and c-jun, proteases, protease inhibitors, including collagenase type I and plasminogen activator inhibitor, and adhesion molecules, including intercellular adhesion molecule I.
  • Protein kinase C activation antagonizes growth factor activity by the rapid phosphorylation of the epidermal growth factor receptor. Phosphorylation decreases tyrosine kinase activity.
  • a set of immediate-early genes are rapidly induced via a pathway that does not require protein synthesis.
  • cytokines include transcriptional factors, cytokines, cytoskeletal proteins, nuclear hormone receptors and extracellular matrix receptors.
  • Cell surface receptors bind circulating signal polypeptides, such as growth factors and hormones, as the initiating step in the induction of numerous intracellular effector functions.
  • Receptors are classified on the basis of the particular type of pathway that is induced. Included among these classes of receptors are those that bind growth factors and have intrinsic tyrosine kinase activity, such as the HBGF receptors and those that couple to effector proteins through guanine nucleotide binding regulatory proteins, hereinafter referred to as G-protein coupled receptors and G- proteins, respectively.
  • G-protein transmembrane signaling pathways consist of three proteins: receptors, G proteins and effectors.
  • G proteins which are the intermediaries in transmembrane signaling pathways, are heterodimer ⁇ and consist of ⁇ , ⁇ and gamma subunits. Among the members of a family of G proteins the ⁇ subunits differ. Functions of G proteins are regulated by the cyclic association of GTP with the ⁇ subunit followed by hydrolysis of GTP to GDP and dissociation of GDP.
  • G-protein coupled receptors are a diverse class of receptors that mediate signal transduction by binding to G- proteins. Signal transduction is initiated via ligand binding to the cell membrane receptor, which stimulates binding of the receptor to the G-protein.
  • the receptor-G-protein interaction releases GDP, which is specifically bound to the G-protein, and permits the binding of GTP, which activates the G-protein.
  • Activated G-protein dissociates -from the receptor and activates the effector protein, which regulates the intracellular levels of specific second messengers.
  • effector proteins include adenylyl cyclase, guanylyl cyclase, phospholipase C, and others.
  • G-protein-coupled receptors which are glycoproteins, are known to share certain structural similarities and homologies (see, e.g. , Gilman, A.G., Ann. Rev. Biochem.56: 615-649 (1987), Strader, CD. et al.
  • G-protein- coupled receptors that have been identified and cloned are the substance K receptor, the angiotensin receptor, the ⁇ - and ⁇ - adrenergic receptors and the serotinin receptors. G-protein- coupled receptors share a conserved structural motif. The general and common structural features of the G-protein- coupled receptors are the existence of seven hydrophobic stretches of about 20-25 amino acids each surrounded by eight hydrophilic regions of variable length.
  • each of the seven hydrophobic regions forms a transmembrane ⁇ helix and the intervening hydrophilic regions form alternately intracellularly and extracellularly exposed loops.
  • the third cytosolic loop between transmembrane domains five and six is the intracellular domain responsible for the interaction with G-protein.
  • G-protein-coupled receptors are known to be inducible. This inducibility was originally described in lower eukaryotes. For example, the cAMP receptor of the cellular slime mold, Dict ⁇ ost--f » ⁇ -imn. is induced during differentiation (Klein et al.. Science 241: 1467-1472 (1988).
  • cAMP During the Dictyostelium t.isnni ⁇ nm differentiation pathway, cAMP, induces high level expression of its G-protein-coupled receptor. This receptor transduces the signal to induce the expression of the other genes involved in chemotaxis, which permits multicellular aggregates to align, organize and form stalks (see, Firtel, R.A. , et al. Cell 58: 235-239 (1989) and Devreotes, P., Science 245: 1054-1058 (1989)).
  • H a n endothelial cells utilize a series of morphological correlates during its differentiation pathway, discussed supra.. in which individual cells migrate, align and organize to form multicellular capillary-like structures.
  • a DNA molecule that encodes edg-1 gene product which is the product of an immediate-early gene that is expressed in the early stage of differentiation of endothelial cells in response to PMA or IL-1.
  • This invention provides a gene and protein, which is the first immediate-early gene that encodes a G-protein-coupled receptor.
  • RNA was isolated.
  • Total RNA (10 ⁇ g) was fractionated by 1% agarose-formaldehyde gel electrophoresis, blotted onto a zeta-probe membrane and hybridized with [ 32 P]-labeled edg-1 (A) or a GAPDH (B) cDNA probes.
  • the following reagents were used: PMA (20 ng/ml) , chx (5 ⁇ g/ml) , Actinomycin D (Act D) (2 ⁇ g/ l) .
  • Act D Actinomycin D
  • FIG. 3 Confluent cultures of HUVEC were pre-treated with 20 ng/ml PMA for 4 hour. Either Act D (2 ⁇ g/) alone or with chx (5 ⁇ g/ml) was added to the cultures, at a time designated 0. At the indicated time points, cultures were harvested and Northern blot analysis was performed on total RNA as described above using the edg-1 (A) and GAPDH (B) cDNA probes.
  • FIG. 4 HUVEC were either untreated or treated with 20 ng/ml PMA for 2 hour after which nuclei were prepared. Run-off transcripts were obtained by labelling 10 7 nuclei in vitro with D2 P]-UTP. RNA was purified and hybridized to immobilized plasmid DNA encoding edg-1 (10 ⁇ g/slot) , human fibronectin (fn) (2 ⁇ g/slot) and pBlues ⁇ ript (pBS) (10 ⁇ g/slot) .
  • Figure 5. Nucleotide and Deduced Amino Acid Sequence of Human edg-1. The nucleotide (1-2774) and deduced amino acid sequence (1-380) is shown for human edg-1 cDNA.
  • the deduced transmembrane domains are underline and potential N-linked glycosylation sites are shown with ann asterisk. Possible serine and threonine phosphorylation sites are shown with closed circles. The basic amino acid-rich intracellular domain, which is located between transmembrane domains five and six is highlighted with open circles. THe Kozak consensus translation initiation sequence (5') and polyadenylation sites (3') are shown with double lines underneath their respective sequences. The Genbank accession number for this nucleotide sequence is M31210.
  • FIG. 6 The amino acid sequence of the putative edg- 1 translation product was aligned with Substance K receptor (SKR) , Substance P receptor (SPR) , S 2 -adrenergic receptor (B2AR) , Serotonin receptor lc (5HTC) , ⁇ 2 -adrenergic receptor (A2A) , Serotonin receptor la (5HTla) , Rhodopsin (OSPD) and angiotensin receptor (MAS) . Highly homologous regions are boxed and indicated on the linear schematic.
  • SSR Substance K receptor
  • SPR Substance P receptor
  • B2AR S 2 -adrenergic receptor
  • Serotonin receptor lc 5HTC
  • A2A Serotonin receptor la
  • OSPD Rhodopsin
  • MAS angiotensin receptor
  • FIG. 7 A structural model for the putative edg-l translation product is shown. This model is analogous to other G-protein-coupled receptors. The potential N-linked glycosylation sites are indicated with an inverted M Y M . Potential phosphorylation sites at serine and threonine residues are shown with dark circles. The third cytosolic intracellular domain, which is between transmembrane domains 5 and 6 contains a highly basic region (11/35 residues) is also indicated.
  • Figure 8. Hydrophobicity Profile of edg-1 Translation Product The deduced amino acid sequence of edg-1 was analyzed for hydrophobic regions and the amino acid sequence (residues) plotted against the hydrophobicity index. The putative transmembrane (TM) domains are indicated.
  • FIG. 9 Expression of edg-1 transcript in human cells.
  • Total RNA (5 ⁇ g) from human saphenous vein smooth muscle cells (S) , foreskin fibroblasts (F) , HeLa cells (H) , epidermoid carcinoma (A431) cells (A) , melanocytes (M) , brain tissue (B) and endothelial cells (E) were reverse transcribed into cDNA and amplified with edg-1 specific oligonu ⁇ leotide primers that span the carboxy-terminal tail domain (A) and the third cytosolic loop (B) .
  • Amplified DNA was separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
  • Molecular weight markers are from top to bottom: 1.6 Kb, 1.0 Kb, 0.5 Kb, 0.4 Kb, 0.3 Kb, 0.2 Kb and 0.15 Kb. It can be seen in (A) that transcript of the expected size, about 600 bp, , which was amplified using oligonu ⁇ leotide primers specific for the C-terminal domain, was present in RNA from all the cultured cell lines and human brain. In contrast, when the transcript was amplified using an a pair oligonucleotides that span the third intracellular loop, cell or tissue specific bands were observed.
  • the edg-G-protein-coupled receptor family is a family of related proteins that share substantial homology and structure and that contain common constant regions or domains but differ in at least one variable region or.domain that includes the third cytosolic loop. See, e.g.. Figures 6, 7, and 9. The particular variable region and, thus, each family member, is expressed in a tissue-specific manner.
  • tissue-specific expression of a transcript in a tissue-specific manner includes expression of transcripts that are expressed in only certain tissues or cell types. Such tissue- specific expression can be effected through a variety of mechanisms, including the expression of different genes in each tissue or cell type, through alternative splicing of the same gene in each tissue or cell type, or through recombination of germ line DNA in during development or differentiation of each cell type.
  • the edg-1-G-coupled protein receptor transcript is the intermediate early transcript that is expressed in the early stage of differentiation in endothelial cells that can be induced or stimulated with PMA and interleukin-1 (IL-1) but not with TGF-,3, HBGF-1, or ⁇ - thrombin.
  • the edg-1 G-coupled protein receptor transcript encodes the edg-1 G-coupled protein receptor.
  • the edg-1-G-coupled protein receptor transcript family is a family of transcripts that are expressed in a tissue-specific manner and encode members of the family of related proteins that share substantial homology and structure and that contain common constant regions or domains but differ in at least one variable region that includes the third cytosolic loop.
  • DNA encoding a prot in includes any DNA
  • the edg-1 transcript refers to the 2.8
  • the edg-1 transcript also refers to this
  • cells may be isolated from any source of endothelial RNA.
  • HUVEC endothelial cells
  • the desired immediate-early genes can be identified by any means in which the transcripts comparing the transcripts in cells that are stimulated with PMA, IL-2 or other indu ⁇ er with the transcripts that are present in untreated cells. Those that are present only in the treated cells are, thus, immediate-early genes.
  • any member of the G- protein-coupled receptor family of this invention can be identified by screening an appropriate library with an appropriate probe derived from the edg-1 clone. For example, an appropriate probe would be one derived from the 3' end of the clone. Any methods known to those of skill in the art to accomplish this may be used.
  • the immediate-early gene of this invention is the edg-1 encoding gene. It is induced by IL-1, LPS or PMA, but not by HBGF-1, TGF-0, or ⁇ -thrombin.
  • the edg- 1 clone provided herein encodes a protein that shares many structural and sequence similarities with known G-protein- coupled receptors, including the 3-adrenergic, substance K, substance P, rhodopsin, serotonin (5-HT) , tachykinin receptors and the cAMP receptor of Dictyostelium.
  • the N-linked glycoslyation site at Asn 30 is also found in the Substance K and angiotensin receptors.
  • the two N-linked glycosylation sites are found withih the amino-terminal domain of all G-protein-coupled receptors.
  • the region in proximity to the second and third hydrophobic domains is highly conserved among all such receptors, including that encoded by edg-1.
  • Asp 130 is known to be absolutely necessary for G-protein; in the edg-1-encoded protein the Asp/Glu-Arg is conserved.
  • the overall sequence similarity between the edg-1 G-protein-coupled receptor of this invention and other such receptor is quite divergent, there is a significant degree of sequence similarity within the carboxy-terminal half, particularly within transmembrane domain seven.
  • the intracellular hydrophilic loop regions contain four potential phosphorylation sites at residues Thr ⁇ , Ser- g .,, Thr- ⁇ g and at Ser 351 .
  • This feature is common to many G-protein- coupled receptors. Phosphorylation at the Ser and Thr residues within the intracellular domains has been implicated in the phenomenon of receptor desensitization.
  • the hydrophilic region between transmembrane domains five and six is the region that is absolutely necessary for G- protein coupling and it is highly divergent among members of the G-protein-coupled receptor proteins. In the G-protein- coupled receptor that is encoded by edg-1, this region is highly basic.
  • the family of edg-1 related tissue-specific proteins provided in this invention differ in this region and, thus, most likely differ in their respective binding or coupling interactions with the G-protein or protein ligands.
  • the ligand that binds to each of the members of the family of G-protein-coupled receptor proteins of this invention can be identified by methods that are known to those of skill in the art. For example, xenopus oocytes can be transfected with DNA that encodes the particular protein. The protein will be expressed on the cell surface of the oocytes. Since these oocytes are sensitive to calcium exchange across the cell membrane, binding of the appropriate ligand causes calcium exchange across membrane. Labeled calcium can be used and the ligand that causes labeled calcium exchange can be identified.
  • ATP ATP
  • AMP adenosine
  • leukotrienes prostenoids
  • histamine histamine
  • bombasin thrombin
  • azopressin bradykinin
  • endothelin endothelin
  • serotensin substance P and neuropeptide.
  • the following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
  • IL-l ⁇ interleukin a ⁇
  • HBGF-l ⁇ Recombinant human HBGF-l ⁇ was obtained from Anthony Jackson, American Red Cross, Rockville, MD.
  • Porcine TGF-3 was purchased from R & D Systems.
  • RNA Preparation and cDNA Library Construction Total RNA was obtained from cells that either untreated or treated with 20 ng/ml PMA (Sigma) and 5 ⁇ g/ml of cycloheximide (hereinafter chx) (Sigma) for 4 hours. The cells were rinsed with phosphate-buffered saline, lysed in 4M guanidinium isothiocyanate and total RNA purified as described in Winkles, J. , et al. ( (1987) Proc. Natl. Acad. Sci. USA 84, 7124-7128) .
  • RNA (10 ⁇ g) from HUVEC exposed to PMA and chx was converted to double-stranded cDNA and cloned into the Eco Rl site of lambda gtlO, using the cDNA synthesis kit from Bethesda Research Labs (Gaithersburg, MD) and the cDNA cloning kit from Amersham (Chicago, IL) .
  • the library contained > 10 6 independent clones, with an average insert size of approximately 11Kb.
  • RNA 10 ⁇ g was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde, capillary-blotted onto Zeta-probe membrane (Biorad) and UV cross-linked (Maniatis et al. (1982) In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • the cDNA insert fragment for edg-1 (2.8 Kb) or human GAPDH (1 Kb) was labeled to high specific activity (>108 cp / ⁇ g) using a random primer labeling kit (BRL) and was used to hybridize filters in Church-Gilbert buffer (0.5 M sodium phosphate pH 7.2, containing 7% SDS and 1% bovine serum albumin, lmM EDTA and 20% formamide at 65° C for 16-20 hrs. Filters were washed twice for 15 min at high-stringency (O.lxSSC, 65° C) .
  • the differential screen was performed by plating 2 x 10 4 pfu of the library onto bacteriological plagues (15 cm diameter) containing LB agar. The phage were allowed to grow at 37° C until plaques were approximately 0.5 mm in diameter. Phage DNA was adsorbed onto Gene-screen plus nylon filters (Dupont, DE) , in duplicate, denatured, neutralized, and UV cross-linked.
  • the probe for differential screening was prepared by reverse transcription of 1 ⁇ g of poly A + RNA from control and PMA/chx-treated HUVEC.
  • the reaction conditions were as follows: 50 mM Tris HC1, pH 8.3, 75 mM KCl, 20 mM dithiothreitol, 3 mM MgCl 2 , 500 20 ⁇ M dCTP, 200 ⁇ M each of dATP, dCTP, and dTTP, 0.5 ⁇ g/ml of oligo dT 12 . 18 and 400 units of MMLV-reverse transcriptase (Bethesda Research Labs, Gaithersburg, MD) . After incubation at 37° C for 60 minutes, RNA was hydrolyzed by treatment with 100 ⁇ l 0.6M NaOH and 20 mM EDTA for 30 minutes at 65° C.
  • the cDNA was purified on Sephadex G-50 columns and ethanol-precipitated.
  • Duplicate filters were incubated with 10 cpm/ml of cDNA for 48 hours at 65° C in hybridization buffer containing 2% SDS, 1 M NaCl and 10% dextran sulfate.
  • the filters were washed twice for 30 min at 65° C with 2xSSC containing, 1% SDS followed by two additional washes for 30 min at 65° C with O.lxSSC containing 1% SDS.
  • the filters were autoradiographed and duplicates were superimposed on each other to isolate PMA/chx-induced signals. Differential signals were plaque-purified by repeating the screening process.
  • Insert cDNA was prepared and used for eith- er Northern blot analysis or subcloning into plasmid vectors. Of the twelve positive signals obtained from >10 5 pfu of the library three were found to be consistently positive. Two of the clones had inserts had sequences identical to the sequence of DNA that encodes human collagenase Type 1. The third clone, herein called edg-1 (1-Kb) contained a poly A tract at 3' end, a unique nucleotide sequence and hybridized to a 3.0 Kb PMA indueible mRNA species. This 1 kb insert was used to rescreen two additional cDNA libraries-lambda gtlO and cDM8.
  • EXAMPLE 4 In order to ascertain at what level PMA induces edg- 1 mRNA, edg 1 induction in the presence of actinomycin D was investigated. As shown in Figure 2, actinomycin D repressed the inductive effect of PMA, which-suggests that PMA induces the transcription of the edg-1 gene.
  • EXAMPLE 5 Nuclear Run-On Transcription. Nuclei (10 7 ) were prepared from quiescent HUVEC untreated or treated with 20 ng/ml PMA for 2 hr.
  • run-off transcripts were prepared by incubating the nuclei with 250 ⁇ Ci of[ ⁇ - 32 P]-UTP (.6000 Cl/mmol, Amersha ) , lOmM ATP, CTP, GTP, in the reaction buffer containing 20mM Tris-HCl, pH 7.9, 140mM KC1, lOmM MgCl 2 and lmM dithiothreitol as described (Nevins, J., (1987) Meth. Enzymol. 152, 234-240).
  • RNA was purified (Winkles, J., supra.) and hybridized to nylon filters containing either 10 ⁇ g of denatured plasmid edg-1 cDNA, 2 ⁇ g of human fibronectin or 10 ⁇ g of pBluescript (Stratagene) .
  • the hybridization and washing conditions were identical to those described for the differential hybridization.
  • Nuclei were prepared from untreated HUVEC or from HUVEC treated with PMA for 2 hours. Labeled run-on transcripts were obtained and hybridized to immobilized plasmid DNA containing the edg-1 insert and to a control plasmid containing fibronectin-encoding DNA or to a Bluescript plasmid ( Figure 4) .
  • Edg-1 transcription was significantly induced in nuclei from the PMA treated HUVEC.
  • EXAMPLE 6 DNA Sequence Analysis. The structure of the edg-1 gene and gene product was elucidated by DNA sequencing of the 2.8 Kb cDNA clone. Plasmid DNA for edg-1 (2.8Kb) was obtained by screening a cDNA library from HUVEC constructed in the vector, cDM8, which was a gift of Brian See, Harvard Medical School) with the (1.6Kb) insert obtained from the cDNA library in lambda gtlO, discussed in Example 1. Double-stranded sequence analysis was performed using the sequenase-2 enzyme (USBC) , following the manufacturer's instructions. Successive primers were synthesized and used to sequence both strands of the cDNA clone.
  • USBC sequenase-2 enzyme
  • the DNA sequence was analyzed by the Intelligenetics Sequence Analysis program. As shown in Figure 5, the complete nucleotide sequence of the edg-1 cDNA clone is 2774 bp long and, at nucleotide 251 from the 5'end, contains a consensus translation initiation sequence, which is followed by an open-reading frame (ORF) that encodes 380 amino acids. The ORF is followed by a 3*, A/T-rich, 1.3 Kb untranslated region followed by a poly A tail. A/T rich sequence motifs in 3' untranslated regions have been implicated in conferring rapid RNA degradation of intermediate-early mRNAs.
  • ORF open-reading frame
  • the edg-1 clone also contains about 250 bp of 5•untranslated region.
  • the deduced amino acid sequence contains a non- hydrophobic amino-terminal stretch of 46 amino acids, which contain two potential N-linked glycosylation sites at residues 29 and 35. This stretch is followed by seven alternating stretches of hydrophobic regions, each about 20 amino acid residues long. There are 8 hydrophilic regions. Each of the hydrophobic regions is flanked by hydrophilic regions of 7 to 19 amino acids, except for the region between the fifth and sixth transmembrane domain, which is 35 residues long and is rich in basic and dibasic residues.
  • RNA from HUVEC was purified as described in Example 1. RNA from human saphenous vein smooth muscle cells, human foreskin fibroblasts, human epidermoid carcinoma cells (A431) , human cervical carcinoma cells (HeLa) , human melanocytes and total brain were the generous gift of Dr. Jeffrey Winkles of the American National Red Cross.
  • RNA total RNA (5 ⁇ g) from all the cultured cells and poly A + RNA (1 ⁇ g) from human brain (Clontech) was converted to cDNA by treatment with 200 units of MMLV reverse transcriptase (Bethesda Research Labs, MD) in 50 mM Tris-HCl, pH, 8.0, 1 mM dithiothreitol, 15 mM NaCl, 3 mM MgCl 2 , 1 unit RNAsin (Promega) , o.2 ⁇ g of random hexamer primers, 0.8 mM dNTPs and incubated for 1 hour at 37° C.
  • MMLV reverse transcriptase Bethesda Research Labs, MD
  • 50 mM Tris-HCl pH, 8.0, 1 mM dithiothreitol, 15 mM NaCl, 3 mM MgCl 2 , 1 unit RNAsin (Promega)
  • the reaction was terminated by heating at 95° C for 10 minutes and diluted to 1 ml with distilled water. Enzymatic amplification was done on a 10 ⁇ l aliquot of the cDNA mix. PCR was performed in 50 mM Tris-HCl, pH, 8.0, 1.5 mM MgCl 2 , 10 mM KC1, 0.2 mM dNTPs, 0.5 ⁇ g each of primers for edg-1 and 2.5 units of Taq DNA polymerase (Cetus, CA) (see, Saiki et al. (1988) Science 239, 487-491).
  • the reaction mixture was heated at 94° C for 1 minute, annealed at 55° C for 2 minutes, and extended at 72° C for 3 minutes for 30 repetitive cycles.
  • the primers used were as follows: (1) 5 « -TG TAC TGC AGA ATC TAC T-3' (sense) and 5'-T GCA GCC CAC ATC CAG CAG CA-3' (antisense) to amplify from nucleotide no. 909 to 1094, which spans the third cytosolic domain; and (2) 5' AAG ACC TGT CAC ATC CTC TTC-3 « (sense) and 5* ATG AAC CCT TTA GGA GCT TGA CAA-3' (antisense) to amplify from nucleotide no.
  • RNA from the various cultured human cell lines and from human brain was reverse transcribed and the cDNAs amplified using the oligonucleotides that are specific for the C-terminal domain (amino acids 266 to the termination codon and 309 bp of the 3' untranslated region, nucleotides 1100 to 1702, see, e.g.. Figures 5-7 and 9) an amplified product is the expected size, 600 bp., is observed (see Fig. 9 (A)) in RNA from all cell types and human brain.
  • the intensity of the signal was most prominent in endothelial cells, but was present to a lesser extent in smooth muscle cells, fibroblasts, epider oid cells, melanocytes, and brain tissue.
  • the cDNAs were amplified with a pair of oligonucleotides that span the third intracellular loop (amino acids 220-282, nucleotides 909-1094)
  • cell-specific bands were amplified ( Figure 9 (B)) .
  • smooth muscle cells a major band at 0.7 Kb and minor bands at 0.9, 0.3, and 0.19 Kb were observed.
  • HeLa cells a very prominent band was observed at 0.3 Kb.
  • the expected 0.19Kb amplification product was observed only in endothelial cells.
  • tissue specific transcripts differ in the region that encodes the portion of the receptor that couples with the G- protein and thereby modulates the cellular response of the particular cell type to the specific signal.

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Abstract

Nouvelle famille de gènes et de protéines spécifiques tissulaires qui sont apparentés à un gène récepteur couplé à une protéine G, et à la protéine réceptrice. Le gène est un gène intermédiaire précoce qui est exprimé dans des cellules endothéliales différenciatrices. La présente invention décrit notamment un gène, edg-1, qui est un gène précoce immédiat qui encode un récepteur couplé à une protéine G dans des cellules endothéliales. Elle décrit aussi la protéine réceptrice couplée à une protéine G, qui est encodée par edg-1.
PCT/US1991/002344 1990-04-05 1991-04-04 Famille de proteines apparentee a une proteine precoce immediate exprimee par des cellules endotheliales humaines durant la differenciation Ceased WO1991015583A1 (fr)

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Cited By (30)

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EP1369430A3 (fr) * 1997-05-07 2004-01-28 Human Genome Sciences, Inc. Deux récepteurs couplés par des protéines G: GPCR2 induit par VEB (EBI-2) et GPCR de type GDE-1
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US8044076B2 (en) 2006-09-21 2011-10-25 Actelion Pharmaceuticals Ltd. Phenyl derivatives and their use as immunomodulators
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WO2014141171A1 (fr) 2013-03-15 2014-09-18 Actelion Pharmaceuticals Ltd Dérivés de pyridin-4-yle
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Publication number Priority date Publication date Assignee Title
US6855550B2 (en) 1990-09-13 2005-02-15 Duke University Expression of G protein coupled receptors in yeast
US5739029A (en) * 1990-09-13 1998-04-14 Duke University Vectors for expression of G protein coupled receptors in yeast
US6168927B1 (en) 1990-09-13 2001-01-02 Duke University Expression of G protein coupled receptors in yeast
US7413876B2 (en) 1990-09-13 2008-08-19 Duke University Office Of Science And Technology Expression of G protein coupled receptors in yeast
WO1997000952A3 (fr) * 1995-06-20 1997-03-20 Incyte Pharma Inc Homologue du recepteur de edg-2 de l'homme
EP1369430A3 (fr) * 1997-05-07 2004-01-28 Human Genome Sciences, Inc. Deux récepteurs couplés par des protéines G: GPCR2 induit par VEB (EBI-2) et GPCR de type GDE-1
US6887683B1 (en) 1997-05-07 2005-05-03 Human Genome Sciences, Inc. Human G-protein coupled receptors
GB2379880A (en) * 1998-01-08 2003-03-26 Microsense Cardiovascular Sys A method for protecting a remotely monitorable sensor during deployment into a bodily lumen.
GB2379880B (en) * 1998-01-08 2003-05-07 Microsense Cardiovascular Sys Method for protecting a remotely monitorable sensor during deployment in a bodily lumen.
EP1070080A4 (fr) * 1998-03-09 2004-12-29 Smithkline Beecham Corp POLYNUCLEOTIDES ET POLYPEPTIDES D'EDG-1c HUMAIN ET LEURS PROCEDES D'UTILISATION
US6423508B1 (en) 1998-03-09 2002-07-23 Smithkline Beecham Corporation Polynucleotide sequences of human EDG-1c
WO2001011022A1 (fr) * 1999-08-05 2001-02-15 Smithkline Beecham Corporation Recepteur edg3 couple a la proteine g des souris
US7521192B2 (en) 2001-04-18 2009-04-21 Rigel Pharmaceuticals, Inc. EDG: modulators of lymphocyte activation and migration
WO2004058149A2 (fr) 2002-12-20 2004-07-15 Merck & Co., Inc. 1-(amino)indanes et (1,2-dihydro-3-amino)-benzofuranes, benzothiophenes et indoles utilises en tant qu'agonistes du recepteur edg
US7750040B2 (en) 2004-07-29 2010-07-06 Actelion Pharmaceuticals Ltd Thiophene derivatives
US8039644B2 (en) 2005-03-23 2011-10-18 Actelion Pharmaceuticals Ltd. Hydrogenated benzo (C) thiophene derivatives as immunomodulators
US7846964B2 (en) 2005-03-23 2010-12-07 Actelion Pharmaceuticals Ltd. Thiophene derivatives as spingosine-1-phosphate-1 receptor agonists
US7605269B2 (en) 2005-03-23 2009-10-20 Actelion Pharmaceuticals Ltd. Thiophene derivatives as Sphingosine-1-phosphate-1 receptor agonists
US7723378B2 (en) 2005-03-23 2010-05-25 Actelion Pharmaceuticals Ltd. Hydrogenated benzo (C) thiophene derivatives as immunomodulators
US7951794B2 (en) 2005-06-24 2011-05-31 Actelion Pharmaceuticals Ltd. Thiophene derivatives
US7981924B2 (en) 2005-11-23 2011-07-19 Actelion Pharmaceuticals Ltd. Thiophene derivatives
US8003800B2 (en) 2006-01-11 2011-08-23 Actelion Pharmaceuticals Ltd. Thiophene derivatives as S1P1/EDG1 receptor agonists
US8178562B2 (en) 2006-01-24 2012-05-15 Actelion Pharmaceuticals, Ltd. Pyridine derivatives
US8697732B2 (en) 2006-01-24 2014-04-15 Actelion Pharmaceuticals Ltd. Pyridine derivatives
US8133910B2 (en) 2006-09-07 2012-03-13 Actelion Pharmaceuticals Ltd. Thiophene derivatives as S1P1/EDGE1 receptor agonists
US8580824B2 (en) 2006-09-07 2013-11-12 Actelion Pharmaceuticals Ltd. Pyridin-4-yl derivatives as immunomodulating agents
US8288554B2 (en) 2006-09-08 2012-10-16 Actelion Pharmaceuticals Ltd. Pyridin-3-yl derivatives as immunomodulating agents
US8044076B2 (en) 2006-09-21 2011-10-25 Actelion Pharmaceuticals Ltd. Phenyl derivatives and their use as immunomodulators
US8592460B2 (en) 2007-03-16 2013-11-26 Actelion Pharmaceuticals Ltd. Amino-pyridine derivatives as S1P1 /EDG1 receptor agonists
US8598208B2 (en) 2007-08-17 2013-12-03 Actelion Pharmaceuticals Ltd. Pyridine derivatives as S1P1/EDG1 receptor modulators
US8299086B2 (en) 2007-11-01 2012-10-30 Actelion Pharmaceuticals Ltd. Pyrimidine derivatives
US8148410B2 (en) 2007-12-10 2012-04-03 Actelion Pharmaceuticals Ltd. Thiophene derivatives as agonists of S1P1/EDG1
US8410151B2 (en) 2008-03-07 2013-04-02 Actelion Pharmaceuticals Ltd Aminomethyl benzene derivatives
US8658675B2 (en) 2009-07-16 2014-02-25 Actelion Pharmaceuticals Ltd. Pyridin-4-yl derivatives
WO2011007324A1 (fr) 2009-07-16 2011-01-20 Actelion Pharmaceuticals Ltd Dérivés pyridin-4-yliques
US8791100B2 (en) 2010-02-02 2014-07-29 Novartis Ag Aryl benzylamine compounds
WO2012098505A1 (fr) 2011-01-19 2012-07-26 Actelion Pharmaceuticals Ltd Dérivés de 2-methoxy-pyridin-4-yl
US9133179B2 (en) 2011-01-19 2015-09-15 Actelion Pharmaceuticals Ltd. 2-methoxy-pyridin-4-yl-derivatives
WO2014141171A1 (fr) 2013-03-15 2014-09-18 Actelion Pharmaceuticals Ltd Dérivés de pyridin-4-yle
US9617250B2 (en) 2013-03-15 2017-04-11 Actelion Pharmaceuticals Ltd. Pyridin-4-yl derivatives
US10385043B2 (en) 2015-05-20 2019-08-20 Idorsia Pharmaceuticals Ltd Crystalline form of the compound (S)-3-{4-[5-(2-cyclopentyl-6-methoxy-pyridin-4-yl)-[1,2,4]oxadiazol-3-yl]-2-ethyl-6-methyl-phenoxy}-propane-1,2-diol
US10836754B2 (en) 2015-05-20 2020-11-17 Idorsia Pharmaceuticals Ltd Crystalline form of the compound (S)-3-{4-[5-(2-cyclopentyl-6-methoxy-pyridin-4-yl)-[1,2,4]oxadiazol-3-yl]-2-ethyl-6-methyl-phenoxy}-propane-1,2-diol
US11390615B2 (en) 2015-05-20 2022-07-19 Idorsia Pharmaceuticals Ltd Crystalline form of the compound (S)-3-{4-[5-(2-cyclopentyl-6-methoxy-pyridin-4-yl)-[1,2,4]oxadiazol-3-yl]-2-ethyl-6-methyl-phenox
US11834443B2 (en) 2015-05-20 2023-12-05 Idorsia Pharmaceuticals Ltd Crystalline form of the compound (s)-3-{4-[5-(2-cyclopentyl-6-methoxy-pyridin-4-yl)-[1,2,4]oxadiazol-3-yl]-2-ethyl-6-methyl-phenoxy}-propane-1,2-diol

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