Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-09
2002-11-26
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S254110, C530S350000, C536S023500
Reexamination Certificate
active
06485922
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for identifying and characterizing compounds that modulate the activity of a member of the EDG family of receptors, including EDG-1, EDG-2, EDG-3, EDG-4, and EDG-5 (gene sequences and encoded amino acid sequences shown in SEQ ID NOS. 4, 1, 5, 6, and 7, respectively), and a similar receptor named PSP-24 (gene sequence and encoded amino acid sequence shown in SEQ ID NOS. 8 and 9).
BACKGROUND
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. Frequently, binding of a ligand to a cell-surface receptor represents the first step in a cascade of events that results in a cellular response. The ligands recognized by specific receptors include a diverse array of molecules such as peptides, deoxyribonucleotide triphosphates and phospholipids.
Research into phospholipid signaling is an area of intense scientific investigation, as more and more bioactive lipids are being identified and their actions characterized. One important addition to the growing list of lipid messengers is lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycero-3-phosphate, LPA), the simplest of all glycerophospholipids. While LPA has long been known as a precursor of phospholipid biosynthesis in both eukaryotic and prokaryotic cells, only recently has LPA emerged as an intercellular signaling molecule that is rapidly produced and released by activated cells, notably platelets, to influence target cells by acting on a specific cell-surface receptor. Moolenaar (1994)
Trends Cell Biol.
4:213-219. Besides being synthesized and processed to more complex phospholipids in the endoplasmic reticulum, LPA can be generated through the hydrolysis of pre-existing phospholipids following cell activation. The best documented example concerns thrombin-activated platelets, where LPA production is followed by its extracellular release. Eichholtz et al. (1993)
Biochem. J.
291:677-680. Platelet LPA is formed, at least in large part, through phospholipase A
2
(PLA
2
)-mediated deacylation of newly generated phosphatidic acid (PA). Gerrard and Robinson (1989)
Biochim. Biophys. Acta
1001:282-285. Distinct PA-specific PLA
2
activity has been identified in platelets (Ca
2+
-dependent) and in rat brain (Ca
2+
-independent), but little is known about its mode of regulation. Billah et al. (1981)
J. Biol. Chem.
256:5399-5403; and Thompson and Clark (1995)
Biochem. J.
306:305-309.
It remains to be examined at what stage of the platelet activation response LPA is produced and how it is released into the extracellular environment. Given the wide variety of LPA responsive cell types, LPA production and release are unlikely to be restricted to platelets. Indeed, there is preliminary evidence that growth factor-stimulated fibroblasts can also produce LPA. Fukami and Takenawa (1992)
J. Biol. Chem.
267:10988-10993. Furthermore, LPA may be formed and released by injured cells, probably due to nonspecific activation of phospholipases. Many other cell systems remain to be examined for LPA production.
In freshly prepared mammalian serum, LPA concentrations are estimated to be in the range of approximately 2-20 &mgr;M, with oleoyl- and palmitoyl-LPA being the predominant species. Tokumura et al. (1994)
Am. J. Physiol.
267:C204-C210; and Eichholtz et al. (1993)
Biochem. J.
291:677-680. LPA is not detectable in platelet-poor plasma, whole blood, or cerebrospinal fluid. Tigyi and Miledi (1992)
J. Biol. Chem.
267:21360-21367. In common with long chain fatty acids, LPA binds with high affinity to serum albumin at a molar ratio of about 3:1. Tigyi et al. (1991)
J. Biol. Chem.
266:20602-20609; Thumser et al. (1994)
Biochem. J.
301:801-806. It is notable that serum albumin contains several other, as yet unidentified lipids (methanol-extractable) with LPA-like biological activity. Tigyi and Miledi (1992)
J. Biol. Chem.
267:21360-21367. This raises the interesting possibility that LPA may belong to a new family of phospholipid mediators showing overlapping biological activities and acting on distinct receptors; conceivably, the ether-linked phospholipid platelet-activating factor (PAF) and the mitogenic lipid sphingosine 1-phosphate may also belong to this putative family. Zhang et al. (1991)
J. Cell Biol.
114:155-167.
The range of biological responses to LPA is quite diverse, ranging from induction of cell proliferation to stimulation of neurite retraction and even slimemold chemotaxis, and the body of knowledge continues to grow as more and more cellular systems are tested for LPA responsiveness. Jalink et al. (1993)
Proc. Natl. Acad. Sci. U.S.A.
90:1857-1861; Jalink et al. (1993)
Cell Growth and Differ.
4:247-255; and Moolenaar (1995)
Curr. Opin. Cell Biol.
7:203-210; Dyer et al. (1992)
Molec. Brain Res.
14:293-301; Dyer et al. (1992)
Molec. Brain Res.
14:302-309; Tigyi and Miledi (1992)
J. Biol. Chem.
267:21360-21367.
Although its precise physiological and pathological functions in vivo remain to be explored, LPA derived from platelets has all the hallmarks of an important mediator of wound healing and tissue regeneration. Thus, in addition to acting as an autocrine stimulator of platelet aggregation, LPA stimulates the growth of fibroblasts, vascular smooth muscle cells, endothelial cells, and keratinocytes. Moolenaar (1994)
Trends Cell Biol.
4:213-219; Jalink et al. (1994)
Biochim. Biophys. Acta
1198:185-196; Van Corven et al. (1989)
Cell
59:45-54; Tigyi et al. (1994)
Proc. Natl. Acad. Sci. U.S.A.
91:1908-1912; Tokumura et al. (1994)
Am. J. Physiol.
267:C204-C210; and Piazza et al. (1995)
Exp. Cell Res.
216:51-64. Intriguingly, it has been observed that LPA acts as an inhibitor of eukaryotic DNA polymerase &agr;. Murakami-Murofushi et al. (1992)
J. Biol. Chem.
267:21512-21517. LPA also exhibits anti-mitogenic activity toward myeloma cells, presumably through a distinct receptor subtype. Tigyi et al. (1994)
Proc. Natl. Acad. Sci.
91:1908-1912; Murakami-Murofushi et al. (1993)
Cell Structure and Function
18:363-370.
In addition to stimulating cell growth and proliferation, LPA promotes cellular tension and cell-surface fibronectin binding, which are important events in wound repair and regeneration. Zhang et al. (1994)
J. Cell Biol.
127:1447-1459; Kolodney et al. (1993)
J. Biol. Chem.
268:23850-23855; and Lapetina et al. (1981)
J. Biochem.
256:5037-5040. As a product of the blood-clotting process, LPA is a normal constituent of serum (but not platelet-poor plasma), where it is present in an albumin-bound form at physiologically relevant concentrations. Tigyi and Miledi (1992)
J. Biol. Chem.
267:21360-21367; and Eichholtz et al. (1993)
Biochem. J.
291:677-680.
Recently, anti-apoptotic activity has also been ascribed to LPA. PCT Application No. PCT/US94/13649. In this study, an actively proliferating cell line was rescued from serum withdrawal-induced apoptosis by LPA. In another study, evidence has been presented suggesting that LPA can suppress apoptosis in vitro as well as in ischemic organs such as heart and liver. Wu et al. (1996)
Transplantation
(in press).
Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation, and infection by human immunodeficiency virus (HIV). Wyllie (1980)
Nature
284:555-556; Kanter et al. (1984)
Biochem. Biophys. Res. Commun.
118:392-399; Duke and Cohen (1986)
Lymphokine Res.
5:289-299; Tomei et al. (1988)
Biochem. Biophys. Res.
Erickson James
Goddard J. Graham
Kiefer Michael
Atairgin Technologies, Inc.
Eyler Yvonne
O'Hara Eileen B.
Orrick Herrington & Sutcliffe LLP
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