Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact.

A purified opioid-binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide-bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet-derived growth factor receptor (PDGF R), the colony-stimulating factor 1 receptor (CSF-1 R) and the c-kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.

Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.

A mu-opioid-specific receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, gel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by NaDodSO4/polyacrylamide gel electrophoresis and was judged to be homogeneous by the following criteria: (i) a single band was detected by autoradiography after NaDodSO4/polyacrylamide gel electrophoresis of 125I-labeled receptor and (ii) the purified preparation had a specific opioid-binding activity of 17,720 pmol/mg of protein, close to the theoretical value. In addition, the Mr 58,000 value agrees closely with that determined by covalently labeling purified receptor with bromoacetyl-[3H]dihydromorphine or with 125I-labeled beta-endorphin and dimethyl suberimidate.

Effects of 5,5'-dithiobis-(2-nitrobenzoic acid) on opiate binding to both the membrane-bound receptor and the partially purified opiate receptor.

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.

Isolation and purification of morphine receptor by affinity chromatography.

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.

Isolation of opiate binding components by affinity chromatography and reconstitution of binding activities.

Rat brain membranes exhibiting stereospecific opiate binding activity were solubilized by sonication and detergent treatment. The active material could be bound to an affinity column containing 6-succinylmorphine but could not be eluted with free agonist. Although two protein peaks could be eluted with NaCl, neither possessed binding activity; however, one of the peaks (A), in combination with an acidic lipid fraction, eluted subsequently from the column, showed stereospecific binding. Opiate ligands of the mu type bound to this protein/lipid mixture with an order of affinities closely correlating with those of the original membrane but one to two orders of magnitude lower; binding of delta, kappa, and sigma prototype opioids was considerably less. The protein/lipid mixture also competed with the membranes for mu ligands. These results suggest that the isolated protein-lipid complex may be a component of the opiate receptor and, specifically, the mu receptor or binding site. However, because of the lower affinities of mu opiates for this complex, it is conceivable that some essential membrane component is still missing. Preliminary analysis of peak A indicates that it contains a broad spectrum of protein bands, but it remains to be seen which of these are essential for activity.

Peptide inhibitor of morphine- and beta-endorphin-induced analgesia.

The synthetic beta-endorphin analogs with the omission of the NH2-terminal [Met]enkephalin segment [beta-endorphin-(6-31) and beta-endorphin-(20-31)] are shown to inhibit morphine- or beta-endorphin-induced analgesia in mice by the tail-flick test, whereas the synthetic NH2-terminal pentadecapeptide beta-endorphin-(1-15) has no inhibitory activity. This study raises the possibility that endogenous inhibiting peptides exist in the brain which play a role in the regulation of endorphin actions.

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.

Alteration of the physicochemical properties of triphosphoinositide by nicotinic ligands.

The concentrations of nicotinic drugs required to effect a 50% transfer of triphosphol[3H]isositide from an aqueous phase to a nonaqueous phase closely approximated their concentrations for both in vivo neuromuscular blocking activity and binding to purified nicotinic receptors, and correlated well (correlation coefficient = 0.95)) with their biological activities measured by other workers in an Electrophorus electroplax preparation. The triphospho[3H]inositide transfer induced by nicotinic ligands was dependent on the lipid concentration and was potentiated by Ca2+. The affinity constants of 45Ca2+ for triphosphoinositide were similar to those for the purified nicotinic receptor. These and other findings suggest the possibility that triphosphoinositide (phosphatidylinositol bisphosphate) is a binding site of the nicotinic receptor.