Mu-subtype opioid receptor

Details Mu-subtype opioid receptor Mu-subtype opioid receptor

1995-04-28

2001-05-01

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, C435S252300, C435S320100, C536S023500

Reexamination Certificate

active

06225080

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to substantially pure opioid receptors.
BACKGROUND OF THE INVENTION
Opioids are a chemically diverse group of compounds which includes naturally occurring peptides and alkaloids as well as a large number of synthetic analogs. The physiological effects of opioid agonists include analgesia, drowsiness, changes in mood, respiratory depression, decreased gastrointestinal motility, nausea, vomiting and alterations in the endocrine and autonomic nervous systems (Jaffe and Martin, in The Pharmacological Basis of Therapeutics, Gilman, A. G. et al., eds.; MacMillan, New York, pages 491-531, 1985).
Opioid Subtypes and Their Receptors
The physiological actions of opioids are mediated through specific receptors that exist in the responsive tissues. In vitro characterization of these receptors by binding of radiolabelled opioid alkaloids to brain membranes was first described in 1973 by three independent groups of investigators was used to search for endogenous opioids in vertebrate brain. This search led to the discovery of met- and leu-enkephalin, two opioid pentapeptides, in 1975. In that same year, other opioid peptides (derived from the gene later designated as “POMC”) were discovered in the pituitary. Selective binding of the different peptide and nonpeptide opioids to the receptors in membranes derived from different tissues and brain regions plus correlations with pharmacological selectivities of the opioid drugs established the existence of opioid receptor subclasses (history reviewed by A. Goldstein in Opioids: Past, Present and Future, Collier, H. O. J. et al., eds.; Taylor and Frances Ltd., London, 1984, pages 127-143).
The best characterized classes of opiate receptors are the mu (&mgr;), delta (&dgr;) and kappa (&kgr;) classes, based on clear differences in their ligand selectivities and pharmacological effects (Lord et al., Nature, 267:495-499, 1977). Sigma (&sgr;) (Jaffe and Martin, supra), and epsilon &egr; (Schulz et al., J. Pharmacol. Exp. Ther. 216:604-606, 1981) are also thought to exist, based on differential pharmacology and ligand binding. There is also evidence for receptor subtypes within these major classes (Jaffe and Martin, supra).
Mechanisms of Action of Opioids
The best characterized effects of opioids on cell metabolism are decreased Ca
2+
conductance, increased K
+
conductance and decreased levels of cAMP (Loh and Smith, Ann. Rev. Pharmacol. Toxicol., 30:123-147, 1990). These functions are among those known to be regulated by the receptor-associated G proteins, which also confer high-affinity ligand binding on the receptors they associate with (Birnbaumer et al., Biochem. Biophys, Acta. 1031:163-224, 1990). In fact, both the binding of opiate agonists and their effects on adenylate cyclase have been shown to be GTP-dependent. It is likely that a thorough understanding of the signaling mechanisms of opiate receptors, including the identification of specific, receptor-associated G proteins, would shed light on underlying the mechanisms of functions such as analgesia and addiction.
Numerous previous attempts have been made to isolate opioid receptors. Bidlack et al. (PNAS U.S.A. 78:636-639, 1981) disclose the isolation of three species in the molecular weight range of 25-50,000 daltons, isolated from rat brain using affinity chromatography with 14-&bgr;-bromoacetamido-morphine. Gioannini et al. (J. Biol. Chem. 260:15117-15121, 1985) describe the isolation of a 65,000 MW protein from bovine striatum using affinity chromatography with &bgr;-naltrexylethylenediamine. Maneckjee et al. PNAS U.S.A. 82:594-598, 1985) disclose three proteins having MWs of 92,000, 42,000 and 35,000, which were identified from rat brain using affinity chromatography with “Hybromet” a &mgr;-selective ligand. Cho et al. (PNAS U.S.A. 83:4138-4142, 1986) and Ueda et al. (Neurosci. Lett. 75:339-344, 1987) both teach the isolation of a 58,000 molecular weight species from rat brain by affinity chromatography with 6-succinyl morphine as ligand. The peptide described by Cho et al. has subsequently been shown not to be a transmembrane spanning protein (Schofield et al., EMBO J. 8:489-495, 1989). Simon et al. (Neuropeptides 10:19-28, 1987) describe the isolation of 65,000 and 58,000 MW peptides from frog brain by affinity chromatography with the opioid peptide DADLE. Ahmed et al. disclose a 66,000 MW species which was isolated from human placenta by binding to thiol-sepharose, followed by gel electrophoresis, and binding to wheat germ agglutinin-agarose. Notwithstanding these many reports, however, none of these species has ever been verified as an opioid receptor, nor has any of them ever been reported to yield either amino acid or nucleotide sequence which was verifiable as encoding a functional receptor. The logical inference is that the “receptors” allegedly purified in these papers were either not adequately pure to permit sequencing, or are not in fact the receptors they were believed to be. A need therefore continues to exist for a verifiable isolated opioid receptor sufficiently pure to allow sequence to be determined. The invention described in the present application now fulfills such a need.
SUMMARY OF THE INVENTION
The present invention relates to a substantially pure opioid receptor protein, and biologically active fragments thereof. In addition the invention relates to the nucleotide sequence encodes that for the opioid receptor. The term “substantially pure” as used throughout the present specification and claims, means a protein free of other non-opioid receptor cellular proteins with which it would normally be associated in its membrane-bound state. Such a protein is essential in order to successfully obtain accurate sequence information. A purified opioid receptor is isolatable by binding a biotinylated opioid ligand with membranes derived from an appropriate tissue source, i.e., one expected to express opioid receptors, to form a receptor:ligand complex. The membranes are then solubilized in a bile-salt like detergent composition, and contacted with an avidin or streptavidin containing affinity substrate, to which the biotinylated receptor:ligand complex will bind. The receptor is eluted from the bound complex by contact with an eluant containing GTP and NaCl or NaCl alone. The eluate is then contacted with a lectin affinity column which specifically binds glycoproteins.
In one embodiment, a receptor is identified by its binding a &bgr;-endorphin ligand. In particular, three species are identifiable by this characteristic in the method described. A primary species has a molecular weight of about 66,000, while two minor species have molecular weights of 140-160,000 and 50-55,000. Based on the affinity for &bgr;-endorphin, and other pharmacological data, these species are believed to represent a &mgr; opioid receptor type.
In addition to its use in sequencing and ultimate cloning of the receptor gene, the purified receptor, or biologically active fragments thereof, can be used in production of monoclonal or polyclonal anti-receptor antibodies and to identify patterns of post translational modifications and to Elucidate associated G. proteins. “Biologically active” in the present context refers not only to fragments which retain ligand binding activity, but also refers to fragments capable of raising an antibody response when injected into a host animal. Such antibodies (poly- or monoclonal) can be used in manipulation. of peripheral opioid receptors involved in gut motility and growth hormone secretion. Such antibodies can also be utilized in drug delivery to specific tissues or for tumor imaging.
Receptor clones isolated utilizing sequence information obtained from the purified protein are useful in identifying other receptor subtypes, in screening for new opioid ligands, and for understanding mechanisms of opioid action, for example, drug addiction.


REFERENCES:
patent: 5242822 (1993-09-01), Marullo et al.
patent: 5389543 (1995-02-01), Bunzow et al.
patent: WO95/19986 (1995-07-01), None
Sambrook et al., p. 16.3

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