title: Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.
abstract: Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University School of Medicine, 3500 N. Broad Street, MERB 851, Philadelphia, PA, 19140, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Chongguang
authors.initials: C
authors.lastname: Chen
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001921:Brain / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D051379:Mice; D011817:Rabbits; D051381:Rats; D017450:Receptors, Opioid, mu / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000906:Antibodies; D017450:Receptors, Opioid, mu