Nucleic acid molecules encoding a KOR3 kappa opioid receptor...

Details Nucleic acid molecules encoding a KOR3 kappa opioid receptor... Nucleic acid molecules encoding a KOR3 kappa opioid receptor...

1997-12-05

2003-12-09

Landsman, Robert S. (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

06660496

ABSTRACT:

BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains. 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 three 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. 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 lacks 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 K 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 a-helical) segments that span the lipid bilayer (Dohlman et al. (1987); Dohlman et al. (1991)).
G proteins consist of three tightly associated subunits, &agr;, &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 humans), 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;&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 rhodopsin in rod cells, which mediates vision in dim light, three highly similar cone pigments mediating color vision have been cloned (Nathans et al. (1986)A; and Nathans et al. (1986)B). All of the family of G protein-coupled receptors appear to be similar to other members of the family of G protein-coupled receptors (e.g., dopaminergic, muscarinic, serotonergic, tachykinin), and each appears to share the characteristic seven-transmembrane segment topography.
When comparing the seven-transmembrane segment receptors with one another, a discernible pattern of amino acid sequence conservation is observed. Transmembrane domains are often the most similar, whereas the amino and carboxyl terminal regions and the cytoplasmic loop connecting transmembrane segments V and VI can be quite divergent (Dohlman et al. (1987)).
Interaction with cytoplasmic polypeptides, such as kinases and G proteins, was predicted to involve the hydrophobic loops connecting the transmembrane domains of the receptor. The challenge, however, has been to determine which features are preserved among the seven-transmembrane segment receptors because of conservation of function, and which divergent features represent structural adaptations to new functions. A number of strategies have been used to test these ideas, including the use of recombinant DNA and gene expression techniques for the construction of substitution and deletion mutants, as well as of hybrid or chimeric receptors (Dohlman et al. (1991)).
With the growing number of receptor sub-types, G-protein subunits, and effectors, characterization of ligand binding and G protein recognition properties of these receptors is an important area for investigation. It has long been known that multiple receptors can couple to a single G protein and, as in the case of epinephrine binding to &bgr;
2
- and &agr;
2
-adrenergic receptors, a single ligand can bind to multiple functionally distinct receptor sub-types. Moreover, G proteins with similar receptor and effector coupling specificities have also been identified. For example, three species of human G
i
have been cloned (Itoh et al. (1988)), and alternate mRNA splicing has been shown to result in multiple variants of G
s
(Kozasa et al. (1988)). Cloning and over production of the muscarinic and &agr;
2
-adrenergic receptors led to the demonstration that a single receptor sub-type, when expressed

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