Characterization of the Rat m3 Muscarinic Acetylcholine Receptor Produced in Insect Cells Infected with Recombinant Baculovirus

Abstract

ThLe 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%) (masshol.). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (= 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 heterogenously glycosylated in the producing insect cells.Keywords. Muscarinic receptor; baculovirus, expression; solubilization; glycosylation; purification.The muscarinic acetylcholine receptors (mAChR) are singlechain polypeptides predicted to traverse the membrane seven times in an a-helical configuration (for review see Hulme et al., 1989Hulme et al., , 1990 Hose:y, 1992). The receptor family modulates a variety of intracellular events (e.g. opening of K' channels, activation or inhibition of enzymes that control levels of intracellular second messengers such as cyclic AMP and inositol trisphosphate) upon agonist activation via the appropriate guanosinenucleotide-binding regulatory proteins (G-protein ; Ashkenazi et al., 1987;Fukuda et al., 1988Fukuda et al., , 1989Pfaffinger et al., 1985;Neher et al., 1988;Yatani et al., 1990). Cloning and nucleotide sequencing have idlentified five distinct subtypes of muscarinic receptors (ml -m5) from mammalian sources Fukuda at al., 1987;Bonner et al., 1987Bonner et al., , 1988Braun et al., 1987; Kubo et ,aL, 1986a,b; Liao et al., 1989; Peralta et al., 1987b). The receptors are not abundant in mammalian organs and their biochemical studies are complicated by the occurence of mixed subtypes in tissues and also cell lines. This makes the accurate identification of subtype-specific agents virtually impossible . To circumvent these prob- Abbreviations. AbhO, 3-(2'-aminobenzhydryloxy)tropane; BCIP, 5-bromo-4-chloro-3-indolyl phosphate p-toluidinium salt ; Con A, concanavalin A ; mAChR, muscarinic acetylcholine receptor; m3, muscarinic acetylcholine recepto:r subtype 3 ; m.o.i., multiplicity of infection; pfu, plaque-forming unit; PhMeSO,F, phenylmethylsulfonyl fluoride; Sf9 cells, Spodopteru frugiperdu insect cells ; WGA, wheatgerm agglutinin; G-protein, guanosine-nucle...

Characterization of δ, κ, and μ Human Opioid Receptors Overexpressed in Baculovirus-infected Insect Cells

“…Previous reports suggested that a portion of recombinant membrane proteins, including G-coupled receptors, produced in Sf9 cells does not reach the cell surface but remains in the endoplasmic reticulum and Golgi compartments because of possible saturation of the translocation machinery of the insect cell (22)(23)(24)(25). This hypothesis is also supported by our immunofluorescence experiments on permeabilized Sf9 cells expressing hMOR-his in which antibody labeling was detected not only at the cell surface but also inside the cell.…”

Oligomeric potential of the M2 muscarinic cholinergic receptor

“…The hydrophilic antagonist N-methylscopolamine afforded little or no protection when added at the time of infection. It therefore appears that the functional oligomers obtained upon treatment with quinuclidinylbenzilate derived largely from intracellular compartments, in accord with the observation that the localization of muscarinic receptors in Sf9 cells is largely intracellular (Vasudevan et al 1995).…”

A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

“…These include the V2 vasopressin receptor (this study, Fig. 3), platelet-activating factor receptor (46), metabotropic glutamate receptor (47), substance P receptor (48), neurokinin-2 receptor (49), the C5a anaphylaxotoxin receptor (50), glucagon receptor (14), the dopamine D1 receptor (43), D2 receptor (44), the 5HT 1B receptor (45), the M2 muscarinic receptor (12), and the M3 muscarinic receptor (52). Therefore, it is clear that receptor-specific determinants for dimerization must exist.…”