Antibodies to the cloned μ-opioid receptor detect various molecular weight forms in areas of mouse brain

Abstract

Polyclonal antibodies directed against the amino-terminal portion of the cloned rat μ-opioid receptor (μOR) were raised in rabbits. The antibodies diminished the specific binding of 125I-Tyr27-β-endorphin-(1-31) (human) and [3H][D-Ala2,N-MePhe4,Gly-ol5]enkephalin, but not that of the δOR-selective ligand [3H][D-Pen2,5]enkephalin, to mouse brain membranes. The intracerebroventricular administration to mice of affinity-purified anti- μOR IgGs impaired the antinociception produced by the μOR agonists [D- Ala2,N-MePhe4,Gly-ol5]enkephalin and morphine and the μ/δOR agonists β- endorphin-(1-31) and [D-Ala2,D-Leu5]enkephalin, when studied 24 hr later in the tailimmersion test. Antinociception produced by the δOR-selective agonists [D-Pen2,5]enkephalin and [D-Ala2]deltorphin II was fully displayed in these mice. Immunoblots of sodium dodecyl sulfate-solubilized membranes from mouse central nervous system regions revealed protein bands of M(r) 43,000, 51,000, and 58,000. Also detected were bands of higher molecular weights, 100,000 and 114,000, which probably corresponded to dimeric forms, because they disappeared after sonication of the solubilized tissues. This immunoreactivity was present in regions of mouse central nervous system and was barely detected in NG 108-15 cells. After treatment of the solubilized material with endoglycosidase F, the antibodies labeled a band of M(r) 43,000, coincident with the weight of the cloned μOR. These results confirm the existence of several molecular forms of the μOR due to glycosylation.