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Antibodies to the cloned mu-opioid receptor detect various molecular weight forms in areas of mouse brain
J Garzon1, J L Juarros, M A Castro
1Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Abstract:
Polyclonal antibodies directed against the amino-terminal portion of the cloned rat mu-opioid receptor (muOR) were raised in rabbits. The antibodies diminished the specific binding of 125I-Tyr27-beta-endorphin-(1-31) (human) and [3H][D-Ala2,N-MePhe4,Gly-ol5]enkephalin, but not that of the delta OR-selective ligand [3H][D-Pen2,5]enkephalin, to mouse brain membranes. The intracerebroventricular administration to mice of affinity-purified anti-muOR IgGs impaired the antinociception produced by the muOR agonists [D-Ala2,N-MePhe4,Gly-ol5]enkephalin and morphine and the mu/delta OR agonists beta-endorphin-(1-31) and [D-Ala2,D-Leu5]enkephalin, when studied 24 hr later in the tail-immersion test. Antinociception produced by the delta OR-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 NG108-15 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 muOR. These results confirm the existence of several molecular forms of the muOR due to glycosylation.
Insights
Polyclonal antibodies targeting the mu-opioid receptor (muOR) blocked pain relief from muOR agonists in mice. This study confirms multiple muOR molecular forms exist due to glycosylation.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- The mu-opioid receptor (muOR) is a key target for pain management.
- Understanding muOR's molecular characteristics is crucial for developing effective analgesics.
- Previous research suggested heterogeneity in muOR forms, but definitive evidence was lacking.
Purpose of the Study:
- To generate and characterize polyclonal antibodies against the mu-opioid receptor (muOR).
- To investigate the role of muOR in antinociception using these antibodies.
- To identify different molecular forms of muOR in the mouse central nervous system.
Main Methods:
- Raised polyclonal antibodies against the amino-terminal portion of the rat muOR.
- Assessed antibody binding to muOR and delta OR (dOR) selective ligands in mouse brain membranes.
- Evaluated the effect of anti-muOR antibodies on antinociception in mice using the tail-immersion test.
- Analyzed muOR molecular forms via immunoblotting and endoglycosidase F treatment.
Main Results:
- The generated antibodies specifically recognized muOR, inhibiting binding of muOR-selective ligands.
- Intracerebroventricular administration of anti-muOR antibodies significantly impaired muOR agonist-induced antinociception.
- Immunoblotting revealed multiple muOR bands (43, 400, 51,000, and 58,000 Da), including potential dimeric forms.
- Endoglycosidase F treatment indicated that the 43,000 Da band represents the core muOR, with higher molecular weights attributed to glycosylation.
Conclusions:
- The study confirms the existence of multiple molecular forms of the mu-opioid receptor.
- These molecular variations are primarily due to differential glycosylation of the muOR.
- The findings provide critical insights into muOR heterogeneity and its functional implications in analgesia.