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
1994-08-19
2001-11-20
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, C435S254110, C435S320100, C435S325000, C536S023500, C536S024310
Reexamination Certificate
active
06319686
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to compositions of and methods for obtaining opioid receptors. The invention relates as well to the DNA sequences encoding opioid receptors, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant opioid receptor polypeptides. The invention includes as well methods for using the isolated, recombinant receptor polypeptides in assays designed to select and improve among candidate substances such as agonists and antagonists of opioid receptors and polypeptides for use in diagnostic, drug design and therapeutic applications.
2. Description of Related Art
Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and can also induce physical dependence (Koob, et al 1992). The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal and immune functions (Olson, et al 1989). Opioids exert their actions by binding to specific membrane-associated receptors located throughout the central and peripheral nervous system (Pert, et al. 1973). The endogenous ligands of these opioid receptors have been identified as a family of more than 20 opioid peptides that derive from the three precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin (Hughes, et al. (1975); Akil, et al. (1984)). Although the opioid peptides belong to a class of molecules distinct from the opioid alkaloids, they share common structural features including a positive charge juxtaposed with an aromatic ring that is required for interaction with the receptor (Bradbury, et al. (1976)).
Pharmacological studies have suggested that there are at least four major classes of opioid receptors, designated &dgr;, &kgr;, &mgr; and &sgr; (Simon 1991; Lutz, et al. 1992). The classes differ in their affinity for various opioid ligands and in their cellular distribution. The different classes of opioid receptors are believed to serve different physiological functions (Olson, et al., 1989; Simon 1991; Lutz & Pfister 1992). However, there is substantial overlap of function as well as of distribution. Biochemical characterization of opioid receptors from many groups reports a molecular mass of ≈60,000 Da for all three subtypes, suggesting that they could be related molecules (Loh, et al. (1990)). Moreover, the similarity between the three receptor subtypes is supported by the isolation of (i) antiidiotypic monoclonal antibodies competing with both &mgr; and &dgr; ligands but not competing with &kgr; ligands (Gramsch, et al. (1988); Coscia, et al. (1991)) and (ii) a monoclonal antibody raised against the purified &mgr; receptor that interacts with both &mgr; and &kgr; receptors (Bero, et al. (1988)).
Opioids are used clinically in the management of pain, but their use is limited by a constellation of undesirable side effects, including respiratory depression, miosis, decreased gastrointestinal motility, sedation, nausea and vomiting (Jaffe et al., (1990)). A concern of the use of opioids in the treatment of chronic pain is their potential for dependence and abuse. Studies suggest the clinical effects of opioids are mediated via a variety of receptors and that the therapeutic effects and the undesirable side effects of opioids are mediated by different receptor (sub)types (Jaffe et al., (1990); Pasternack, (1993)). Therefore, the therapeutic and side effects of opioids can be separated with the use of more selective agents for receptor subtypes. The present invention discloses the pharmacological properties of the cloned &kgr;, &dgr;, and &mgr; opioid receptors and receptor selectivity of widely employed opioid ligands.
The &dgr; receptors bind with the greatest affinity to enkephalins and have a more discrete distribution in the brain than either &mgr; or &kgr; receptors, with high concentrations in the basal ganglia and limbic regions. Although morphine interacts principally with &mgr; receptors, peripheral administration of this opioid induces release of enkephalins (Bertolucci, et al. (1992)). Thus, enkephalins may mediate part of the physiological response to morphine, presumably by interacting with &dgr; receptors. Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase K
+
conductance, and inactivate Ca
2+
channels through a pertussis toxin-sensitive mechanism (Puttfarcken, et al. 1988; Attali, et al. 1989; Hsia, et al., 1984). These results and others suggest that opioid receptors belong to the large family of cell surface receptors that signal through G proteins (Di Chiara, et al. (1992); Loh, et al. (1990)).
Several attempts to clone cDNAs encoding opioid receptors have been reported. A cDNA encoding an opioid-binding protein (OBCAM) with &mgr; selectivity was isolated (Schofield, et al. (1989)), but the predicted protein lacked transmembrane domains, presumed necessary for signal transduction. More recently, the isolation of another cDNA was reported, which was obtained by expression cloning (Xie, et al. (1992)). The deduced protein sequence displays seven putative transmembrane domains and is very similar to the human neuromedin &kgr; receptor. However, the affinity of opioid ligands for this receptor expressed in COS cells is two orders of magnitude below the expected value, and no subtype selectivity can be shown.
Many cell surface receptor/transmembrane systems consist of at least three membrane-bound polypeptide components: (a) a cell-surface receptor; (b) an effector, such as an ion channel or the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding regulatory polypeptide or G protein, that is coupled to both the receptor and its effector.
G protein-coupled receptors mediate the actions of extracellular signals as diverse as light, odorants, peptide hormones and neurotransmitters. Such receptors have been identified in organisms as evolutionarily divergent as yeast and man. Nearly all G protein-coupled receptors bear sequence similarities with one another, and it is thought that all share a similar topological motif consisting of seven hydrophobic (and potentially &agr;-helical) segments that span the lipid bilayer (Dohlman et al. 1987; Dohlman et al. 1991).
G proteins consist of three tightly associated subunits, a, &bgr; and &ggr; (1:1:1) in order of decreasing mass. Following agonist binding to the receptor, a conformational change is transmitted to the G protein, which causes the G&agr;-subunit to exchange a bound GDP for GTP and to dissociate from the &bgr;&ggr;-subunits. The GTP-bound form of the &agr;-subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G protein molecules, and from the stimulation by G&agr;-GTP of many catalytic cycles of the effector.
The family of regulatory G proteins comprises a multiplicity of different &agr;-subunits (greater than twenty in man), which associate with a smaller pool of &bgr;- and &ggr;-subunits (greater than four each) (Strothman and Simon 1991). Thus, it is anticipated that differences in the &agr;-subunits probably distinguish the various G protein oligomers, although the targeting or function of the various &agr;-subunits might also depend on the &bgr; and &ggr; subunits with which they associate (Strothman and Simon 1991).
Improvements in cell culture and in pharmacological methods, and more recently, use of molecular cloning and gene expression techniques, have led to the identification and characterization of many seven-transmembrane segment receptors, including new sub-types and sub-sub-types of previously identified receptors. The &agr;
1
and &agr;
2
-adrenergic receptors, once thought to each consist of single receptor species, are now known to each be encoded by at least three distinct genes (Kobilka et al. 1987; Regan et al. 1988; Cotecchia et al. 1988; Lomasney 1990). In addition to rhodopsi
Bell Graeme I.
Reisine Terry
Yasuda Kazuki
ARCH Development Corporation
Fulbright & Jaworski LLP
Kunz Gary L.
Landsman Robert S.
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