Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-02-28
2004-03-23
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S069100, C435S325000, C435S173300, C435S252300, C536S024300, C536S023100
Reexamination Certificate
active
06709830
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for modulating the signalling of a G protein-coupled receptor which is expressed predominantly in lymphoid cells, and influences diverse physiological processes including cell proliferation, autoimmunity and inflammation.
BACKGROUND OF THE INVENTION
Bioactive lysophosphohipids regulate a wide variety of cellular activities including proliferation, smooth muscle contraction, wound healing, tumour cell invasiveness and inflammation. Formation of lysophospholipids is enhanced during oxidation of low density lipoprotein (LDL) and under inflammatory conditions. Most notably, lysophosphatidylcholine (LPC) plays a major aetiological role in atheresclerosis (Lusis, AJ, Nature, 407: pp233-241, 2000), and is also implicated in the pathogenesis of the autoimmune disease Systemic Lupus Erythematosus (SLE) (Wu, R, et al, Lupus, 8: pp142-150, 1999) (Wu, R, et al, Clin Exp Immunol, 115: pp561-566, 1999). Both these diseases can be regarded as chronic inflammatory conditions and oxidatively modified phospholipids are increasingly recognised as autoantigens instrumental in their initiation and progression (Romero, FI, et al, Lupus, 9: pp 206-209, 2000) (Iuliano, L, et al, Blood, 90: pp3931-3935, 1997) (Koh, JS, et al, J Immunol, 165: pp4190-4201, 2000).
LPC is produced by the action of phospholipase A
2
(PLA
2
) on phosphatidylcholine and promotes inflammatory effects including upregulation of endothelial cell adhesion molecules and growth factors (Kume, N, et al, J Clin Invest, 90: pp1138-1144, 1992) (Kume, N, et al, J Clin Invest, 93: pp907-911, 1994), chemotaxis of monocytes (Quinn, MT, et al, PNAS, 84: pp2995-2998, 1994), and stimulation of macrophage activation (Yamamoto, N, et al, J Immunol, 147: pp273-280, 1991). Although its mechanisms of action are poorly understood, LPC exerts both stimulatory and inhibitory effects upon several intracellular signalling molecules in certain contexts, supporting a role for LPC as an intracellular second messenger (Prokazova, NV, et al, Biochemistry (Moscow), 63: pp31-37, 1998) (Nishizuka, T, Science, 258: pp607-614, 1992) (Flavahan, NA, Am J Physiol, 264: ppH722-H727, 1993) (Okajima, F, et al, Biochem J, 336: pp491-500, 1998).
Unlike other lysophospholipids, it was thought that LPC actions were not mediated through specific cellular receptors such as membrane-bound GPCRs, a view that arose from the cell lytic properties of extracellular LPC and its abundance in cell membranes and body fluids (Lee, MJ, et al, Science, 279: pp1552-1555, 1998) (Okajima, F, et al, Biochemical Journal, 336: pp491-500, 1998) (Okita, M, et al, Int J Cancer, 71: pp31-34, 1997). Although studies demonstrating G protein-dependent cellular responses to LPC support the notion that this lysophospholipid also elicits biological effects via one or mote of the 250 plus members within the family of G protein-coupled receptors (Yuan, Y, et al, J Biol Chem, 271: pp27090-27098, 1996) (Okajima, F, et al, Biochemical Journal, 336: pp491-500, 1998), no specific high affinity LPC receptor has yet been identified.
Interest in both LPC and the GPCR family of receptors continues to increase due to data which suggests that they may targets for new diagnostic and therapeutic modalities. For example, from the perspective of their clinical significance, there is considerable focus on GPCRs expressed in the hematopoietic and lymphoid systems as many have been shown to play pivotal roles in the regulation of hematopoiesis and immune function. Receptor/ligand relationships within the GPCR family exhibit significant promiscuity, with many receptors recognizing more than one ligand and vice versa. This is especially true among chemokine receptors. Therefore, a major goal in the art is to identify receptor/ligand interactions within this family of GPCRs, particularly among lymphoid expressed orphan receptors of unknown function.
Consequently, there is a need in the art for the identification of both the receptors for LPC and related molecules as well as the ligands for orphan G protein-coupled receptors. The invention provided herein satisfies this need.
SUMMARY OF THE INVENTION
The invention provided herein describes ligands and methods for modulating a novel G protein-coupled receptor (GPCR), called G2A, a lymphocyte expressed orphan G protein-coupled receptor whose genetic ablation results in the development of autoimmunity. For example, the present disclosure teaches that lysophosphatidylcholine (LPC) is a high affinity ligand for G2A and that sphingosylphosphorylcholine (SPC) is a lower affinity ligand for G2A. In G2A-transfected cells, LPC binds to G2A with high affinity (Kd=65 nM) and specificity, and transiently increases intracellular calcium concentration. In addition, the specific binding of LPC to G2A also induces ERK MAP kinase activation and causes G2A receptor internalisation.
The invention described herein has a number of embodiments. A typical embodiment is a method of modulating G2A receptor mediated signaling in a cell comprising altering the concentration of G2A ligand present in the cell's environment that is capable of binding to and activating the G2A receptor. A specific embodiment consists of a method of modulating LPC mediated activation of a G2A receptor in a cell comprising altering the concentration of LPC capable of binding to and activating the G2A receptor. Yet another embodiment consists of a method of modulating SPC mediated activation of a G2A receptor in a cell comprising altering the concentration of SPC capable of binding to and activating the G2A receptor
Methods that modulate G2A activation can be assessed by a variety of protocols.
In one embodiment, G2A receptor mediated signaling is measured by observing a transient elevation in the concentration of intracellular calcium ([Ca
2+
]). In another embodiment, G2A receptor mediated signaling is measured by observing an induction of ERK MAP kinase activation. In a related embodiment, ligand binding to the G2A receptor is measured by a radioligand binding assay.
The concentration of G2A ligand present in the cell's environment that is capable of binding to and activating the G2A receptor can be altered in a variety of ways. Typically, the concentration of G2A ligand present in the cell's environment that is capable of binding to and activating the G2A receptor is increased by introducing exogenous LPC or SPC into the cell's environment. Alternatively, the concentration of G2A ligand present in the cell's environment that is capable of binding to and activating the G2A receptor is decreased, for example by antibody inhibition of the ligand-receptor interaction or by the LPC antagonist choline. Alternatively, other competitive molecules can be employed in such methods. For example, the concentration of SPC capable of binding to and activating the G2A receptor can be decreased by introducing exogenous LPC into the cell's environment. Alternatively, the concentration of LPC capable of binding to and activating the G2A receptor can be decreased by introducing exogenous SPC into the cell's environment.
REFERENCES:
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I.F. Zohn et al., 2000, “G2A is an oncogenic G protein-coupled receptor,” Oncogene (2000) 19:3866-3877, 2000.
W.H. Moolenaar, 1999, “Bioactive Lysophospholipids and Their G Protein-Coupled Receptors,” Experimental Cell Research 253:230-238, 1999.
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R.J. Davis, “Transcriptional Regulation by MAP Kinases,” Molecular Reproduction and Development, 42:459-467, 1995.
Deng et al., “Identification of a major co-rece
Kabarowski Janusz H. S.
Le Lu Q.
Weng Zhigang
Witte Owen N.
Xu Yan
Basi Nirmals
Eyler Yvonne
Gates & Cooper LLP
The Regents of the University of California
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