Nucleic acid encoding mammalian mu opioid receptor

Details Nucleic acid encoding mammalian mu opioid receptor Nucleic acid encoding mammalian mu opioid receptor

1993-09-13

2001-05-22

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...

C435S242000, C435S320100, C536S023100

Reexamination Certificate

active

06235496

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to compositions of and methods for obtaining mu opioid receptors. The invention relates as well to the DNA sequences encoding mu opioid receptors, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant mu 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 mu opioid receptors and polypeptides for use in diagnostic, drug design and therapeutic applications.
BACKGROUND OF THE INVENTION
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 numerous classes of opioid receptors, including those designated &dgr;, &kgr;, and &mgr; (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 and 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) anti-idiotypic 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).
Morphine interacts principally with &mgr; receptors and peripheral administration of this opioid induces release of enkephalins (Bertolucci et al., 1992). 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. 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 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 &agr;-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 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;&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., 1986A; and Nathans et al., 1986B). 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, muscaric, serotonergic, tachykinin, etc.), 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

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