Characterization of the rat m3 muscarinic acetylcholine receptor produced in insect cells infected with recombinant baculovirus.
The m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy-terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8% (mass/vol). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (approximately 10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE-Sephacel chromatography and lectin-affinity chromatography it can be deduced that the receptor produced in insect cells is heterogeneously glycosylated in the producing insect cells.
Department of Molecular Sciences, James Cook University, Townsville, Australia.
S
S
Vasudevan
E C
EC
Hulme
M
M
Bach
W
W
Haase
J
J
Pavia
H
H
Reiländer
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018411:Spodoptera
D000345:Affinity Labels; D011976:Receptors, Muscarinic