Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-22
2002-10-29
Romeo, David S. (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06472173
ABSTRACT:
1. FIELD OF THE INVENTION
This invention relates in general to novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and fetal liver and to polypeptides encoded by these polynucleotides. In particular, the invention relates to a human chemokine receptor that is a member of a family of G protein-coupled receptors characterized by seven transmembrane domains.
2. BACKGROUND
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve GTP-binding proteins (termed “G proteins”) and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Examples include “G protein-coupled receptors,” such as rhodopsin and the receptors for the adrenergic ligands and dopamine (Kobilka, B. K., et al., PNAS 84:46-50 (1987); Kobilka, B. K., et al., Science 238:650-656 (1987); Bunzow, J. R. et al., Nature 336:783-787 (1988)); G proteins themselves; effector proteins, e.g., phospholipase C, adenylyl cyclase, and phosphodiesterase; and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).
In the inactive state, the membrane-associated G protein is a heterotrimer of alpha, beta, and gamma subunits in which the alpha subunit is bound to GDP. The binding of a ligand to a G protein-coupled receptor stimulates a receptor-G protein interaction that results in the exchange of GDP for GTP on the alpha subunit. The alpha subunit then dissociates from the beta-gamma subunits and interacts with an effector. In the case of epinephrine, for example, a G protein couples the beta-adrenergic receptor to adenylyl cyclase, stimulating the production of cAMP. The resultant rise in cAMP levels activates the cAMP-dependent protein kinase A, which phosphorylates and activates glycogen phosphorylase kinase. The latter, in turn, phosphorylates glycogen phosphorylase, producing a characteristic hormone-stimulated increase in enzymatic activity. Hydrolysis of GTP to GDP, catalyzed by the G protein itself, returns the G protein to its basal, inactive form. Thus, the G protein serves a dual role in signal transduction, namely, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
G protein-coupled receptors mediate the actions of a wide variety of extracellular signals including light, odorants, peptide hormones, and neurotransmitters and have been identified in organisms as evolutionarily divergent as yeast and humans. (See generally, Dolman et al., Ann. Rev. Biochem. 60:653-699 (1991).) Many G protein-coupled receptors share a similar topological motif consisting of seven hydrophobic (and potentially alpha-helical) regions predicted to span the lipid bilayer. Indeed, the transmembrane domains of such receptors are generally the most highly conserved regions of the proteins. Structure-function analyses carried out on the &agr;
2
-, &bgr;
1
-, and &bgr;
2
-adrenergic receptors have shown that the fourth transmembrane segment (TMS IV) contains domains that contribute to the binding selectivity of various agonists and antagonists. TMS VII was shown modulate binding to receptor antagonists. Domains responsible for G protein binding are found the region of the protein encompassing TMS V and TMS VI along with the connecting cytoplasmic loop. These studies also implicate the cytoplasmic loops near TMS's V-VII as determinants of G protein coupling and specificity.
Many 7-TMS receptors include a number of conserved cysteine residues. In both rhodopsin and the &bgr;
2
-adrenergic receptor, cysteines located in the C-terminal domain distal to TMS VII are covalently modified by palmitoylation. The &bgr;
2
-adrenergic receptor has been shown to contain two disulfide bonds that are required for normal ligand binding. Site-directed mutagenesis has revealed that four cysteines are essential for proper cell surface expression and ligand binding. Similar studies of rhodopsin indicate that, as in the &bgr;
2
-adrenergic receptor, a disulfide bond in a hydrophilic loop is critical for directing and/or stabilizing interactions that form the ligand binding domain. By contrast, the yeast &agr;-factor receptor has only two cysteine residues, both of which may be replaced by site-directed mutagenesis without any adverse effect on receptor function.
7-TMS receptors also share the ability to become desensitized, a process by which the receptors become refractory to further stimulation after an initial response, despite the continued presence of the original stimulus. Desensitization results from a reduction in the number of cell surface receptors or from an attenuation of the interaction between the receptor and the G protein (i.e., receptor-G protein “uncoupling”). Many G protein-coupled receptors of the seven-transmembrane-segment class (hereafter “7-TMS receptors”) are rapidly uncoupled after exposure to agonists, a process regulated, at least in part, by phosphorylation.
The 7-TMS receptor family includes receptors for members of the chemokine family of inflammatory cytokines, such as interleukin-8 (hereafter IL-8). The name “chemokine” is derived from the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines comprise the main attractants for inflammatory cells during inflammatory and immune responses. See generally, Baggiolini et al., Advances in Immunology, 55:97-179 (1994). Chemokines have been shown to recruit a wide range of leukocytes to sites of infection, inflammation, and disease. For example, chemokines have been shown to be directly involved in the inflammatory process associated with conditions such as allergies (J Clin Invest Oct. 1, 1997; 100(7):1657-1666 Teixeira MM et al.), asthma (J Immunol Nov. 1, 1997;159(9):4593-4601 Lamkhioued B, et al.), arthritis (J Exp Med Jul. 7, 1997;186(1):131-137 Gong J H et al.), gastric inflammation (Physiol Pharmacol September 1997; 48 (3):405-413 Watanabe N et al.), injury (Eur J Neurosci Jul. 9, 1997;9(7): 1422-1438 Bartholdi D, Schwab M E), transplantation rejection (Transplantation Jun. 27, 1997;63(12):1807-1812 Fairchild R L et al.) and autoimmune disorders (J Neuroimmunol July 1997;77(1):17-26 Miyagishi R et al).
Members of the chemokine family generally exhibit 20-70% amino acid identity to one another and contain several highly-conserved cysteine residues. Chemokines can be classified into various subclasses or subfamilies by virtue of the position and spacing of a set of conserved cysteines, designated C-X-C (e.g., IL-8), C-C (e.g., RANTES) and C (e.g., lymphotactin). The C-X-C subfamily has the first two conserved cysteines separated by one amino acid, and the genes encoding the C-X-C subfamily are predominantly located on human chromosome 4. The C-C subfamily has two adjacent cysteines, and the genes encoding the C-C subfamily are predominantly located on human chromosome 17. The C subfamily has one of the first two conserved cysteines, and the genes encoding the C subfamily are predominantly located on human chromosome 17.
C-X-C chemokines IL-8, GRO&agr;, GRO&bgr;, GRO&ggr;, ENA-78, NAP-2, PF4, and &ggr;IP10 form a subfamily of neutrophil chemoattractants. IL-8, GRO&agr;, and NAP-2 have been shown to compete for similar binding sites on neutrophils and elicit similar biological effects. At least two IL-8 receptors have been characterized, and their genes have been mapped to chromosome 2q34-35. One, designated IL-8 receptor type I, has been shown to bind GRO&agr; and NAP-2, in addition to IL-8. IL-8 acts on neutrophils to induce chemotaxis, hydrogen peroxide production, and exocytosis of intracellular granules, which is associated with an increase in the number of certain neutrophil receptors, such as the CR3 (C3bi) adhesion receptor. IL-8 also induces chemotaxis in basophils and lymphocytes, T lymphocytes, in particular. GRO&agr; and NAP-2 elicit effects in neutrophils similar to those elicited by IL-8.
3. SUMMARY OF THE INVENTION
The invention is based on polynucleotides isolated from cDNA
Ford John
Yeung George
Hyseq Inc
Murphy Joseph F.
Romeo David S.
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