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
1999-03-04
2002-11-12
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S471000, C536S023500
Reexamination Certificate
active
06479256
ABSTRACT:
BACKGROUND OF THE INVENTION
G-protein coupled receptors (GPCRs) are proteins that interact with G-proteins to transmit an intracellular signal. Upon ligand binding, GPCRs trigger the hydrolysis of GTP to GDP by G-protein subunits; GTP hydrolysis is accompanied by a switch from activity to inactivity. It is estimated that there are roughly 1,000 GPCRs [Clapham,
Nature
379:297-299 (1996)] and all characterized to date include a seven transmembrane domain that anchors the receptor to the cell. GPCRs include receptors for opiates, adrenaline, histamine, polypeptide hormones, and photons, among other ligands. These receptors are coupled to a wide variety of cellular second messenger pathways including, for example, pathways that alter intracellular calcium concentrations and cAMP levels.
Among the various GPCRs identified, CD97 appears to be representative of a sub-family of proteins which effect cellular adhesion [McKnight, et al.,
Immunol Today
17:283-287(1996)]. CD97 and related receptors are unique in that their structure includes a transmembrane domain that directly links a cytoplasmic domain that participates in GTP hydrolysis with extracellular protein binding domains that specifically participate in cell-cell adhesion. The extracellular, amino terminal region of CD97 includes numerous cell-cell adhesive motifs, including multiple epidermal growth factor-like (EGF-like) repeats and an integrin binding site [Hamann J, et al., Immunol 155:1942—1950 (1996); Gray, et al.,
J. Immunol
157:5438-5447 (1996)]. Proteins that contain EGF-like repeats have been shown to be involved in cell adhesion events [Campbell, et al.,
Curr. Opin. Struct. Biol.
3:385-392 (1993); Rao,etal.,
Cell
82:131-141 (1995)], and consistent with this observation, heterologous expression of CD97 in COS cells elicits homotypic cell aggregation that can be blocked in the presence of anti-CD97 monoclonal antibodies [Hamann, et al.,
J. Exp. Med.
184:1185-1189 (1996)]. CD97 and related proteins have been referred to as the EGF-7TM subfamily of seven transmembrane receptors [McKNight and Gordon,
Immunol. Today
17:283-2887 (1996)]. Ligands identified for CD97 include members of the integrin family of cell surface adhesion receptors. Various integlins recognize and interact with their cognate ligands through a trimeric amino acid sequence of arginine-glycine-aspartic acid (denoted RGD in the single letter amino acid code) [D'Souza, etal.,
Trends Biochem. Sci.,
16:246-250 (1991)] and this sequence has been identified in the extracellular region of CD97, between the EGF-like repeats and the transmembrane domain.
CD97 has been shown to undergo post-translational proteolytic processing which results in an extracellular (and potentially soluble) alpha subunit and a smaller, integral membrane beta subunit [Gray, et al.,
J. Immunol.
157:5438-5447 (1996)]. The two subunits are associated in a non-covalent manner and the alpha subunit is held at the cell surface through its interaction with the beta subunit. The role of proteolysis is unclear, but it may be a mechanism for receptor down-regulation which is common among proteins, such as selectins and intercellular adhesion molecules (ICAMs), that participate in cell adhesion.
Other members of the CD97 sub-family of GPCRs have been identified by amino acid sequence and structural homology and include human EMR1, HE6, BAI1, the calcium-independent receptor of latrotoxin (CIRL), latrophilin, and proteins encoded by the
Caenorhabditis elegans
open reading frames designated B0457.1 and B0286.2 [Baud V, et al.,
Genomics
26:334-344 (1995); McKnight, et al.,
J. Biol. Chem.
271:486-489 (1996); Krasnoperov, et al.,
Neuron
18:925-937 (1997); Lelianova, et al.,
J. Biol. Chem.
272:21504-21508 (1997); Davletov, et al.,
J. Biol. Chem.
271:23239-23245 (1996); Nishimori, et al.,
Oncogene
15:2145-2150 (1997)]. EMR1, and its murine homolog F4/80, are macrophage-specific in expression and structurally related to CD97 in that they contain multiple extracellular EGF-like repeats, a rod-like stalk region, and the characteristic transmembrane domain of GPCRs [Baud V, et al.,
Genomics
26:334-344 (1995); McKnight, et al.,
J. Biol. Chem.
271:486-489 (1996)]. No ligands have been identified for EMR-1 and it is uncertain if the protein undergoes post-translational proteolytic processing.
CIRL [Krasnoperov, et al.,
Neuron
18:925-937 (1997); Lelianova, et al.,
J. Biol. Chem.
272:21504-21508 (1997); Davletov, etal.,
J. Biol. Chem.
271:23239-23245 (1996)] is believed to be expressed specifically in the central nervous system at neuronal presynaptic terminals and, like CD97, undergoes proteolytic cleavage resulting in an extracellular alpha subunit in non-covalent association with an integral membrane seven-transmembrane beta subunit. Cleavage of latrophilin is believed to occur at a Ser-His-Leu/Thr-Asn-Phe site that is conserved in CD97 [Krasnoperov, et al.,
Neuron
18:925-937 (1997)]. CIRL has been shown to bind latrotoxin, a component of black widow spider venom, in the 0.5 to 1.0 nM range, and binding of the ligand to CIRL expressed in bovine chromaffin cells has been shown to result in exocytosis, a hallmark of toxin binding [Krasnoperov, et al.,
Neuron
18:925-937 (1997)]. Alpha latrotoxin binding has also been demonstrated at neuromuscular motor endplates, and this interaction elicits explosive secretory granule release of acetylcholine from presynaptic granules, resulting in muscle paralysis characteristic of the spider's bite [Petrenko, et al.,
F.E.B.S. Letts.
325:81-85 (1993)]. It is unclear, however, if the peripheral toxin effects result from binding to CIRL or some other related protein.
Thus there exists a need in the art to identify and characterize other members of the CD97-like family of GPCRs, in particular human receptors which participate in cellular adhesion and those that participate in cytoplasmic metabolic pathways modulated by extracellular signals. Identification of CD97-like receptors can permit identification and diagnosis of disease states which arise from aberrant signaling by the receptor, as well as disease states that arise from aberrant expression of the receptor itself.
SUMMARY OF THE INVENTION
The present invention provides purified and isolated human seven transmembrane receptor lectomedin polypeptides or fragments thereof, said polypeptides comprising extracellular lectin-binding, olfactomedin-like, and mucin-like domains. Mature lectomedin polypeptides are also provided wherein signal or leader sequences are cleaved. Preferred polypeptides of the invention comprise the amino acid sequence set out in SEQ ID NO: 2 or a fragment thereof, the amino acid sequence set out in SEQ ID NO: 4 or fragment thereof the amino acid sequence set out in SEQ ID NO: 6 or fragment thereof, and the amino acid sequence set out in SEQ ID NO: 58 or fragment thereof.
The invention also provides polynucleotides encoding polypeptides of the invention. Preferred polynucleotides comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 57. The invention also provides polynucleotides a) encoding a human lectomedin polypeptide selected from the group consisting of the polynucleotide set out in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 57; b) a DNA which hybridizes under moderately stringent conditions to the non-coding strand of the polynucleotide of (a); and c) a DNA which would hybridize to the non-coding strand of the polynucleotide of (a) but for the redundancy of the genetic code. Preferred polynucleotides of the invention are DNA molecules. Preferred DNA molecules are cDNA molecules and genomic DNA molecules. The invention also provides DNA which is a wholly or partially chemically synthesized. Anti-sense polynucleotide which specifically hybridizes with a polynucleotide of the invention are also comprehended.
The invention also proved expression construct
ICOS Corporation
Kunz Gary L.
Landsman Robert S.
Marshall Gerstein & Borun
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