Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells

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Abstract

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin–cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin–cholate, n-dodecyl-β-d-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin–cholate was unaffected by preincubation of the extract for up to 60 min at 30°C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

Introduction

It is commonly held that a G protein, or a subunit thereof, shuttles between its receptor on the one hand and the effector on the other De Lean et al., 1980, Gilman, 1987, Conklin and Bourne, 1993. The receptor therefore is thought to act catalytically, visiting and activating multiple G proteins in succession. Central to this view is the notion that a reversible interaction between receptor and G protein accounts for the GTP-sensitive heterogeneity seen in the binding of agonists De Lean et al., 1980, Ehlert, 1985. Mechanistic proposals along such lines typically embody several postulates, two of which are of interest here. First, heterogeneity is induced by the G protein in a population of mutually independent and otherwise identical sites; second, the transient receptor-G protein complex forms and dissociates spontaneously and rapidly on the time-scale of a binding assay.

Although attractive on many counts, the idea of a transient complex between receptor and G protein becomes problematic when applied in a quantitative and mechanistically consistent manner (e.g., Neubig et al., 1985, Lee et al., 1986, Wong et al., 1986, Wreggett, 1987, Graeser and Neubig, 1993, Neubig, 1994, Green et al., 1997, Wenzel-Seifert et al., 1999). Various lines of evidence suggest that the problem lies in the postulates noted above (Chidiac et al., 1997). In particular, a number of otherwise puzzling effects revealed in the binding properties can be explained in terms of cooperativity among interacting sites (e.g., Henis and Sokolovsky, 1983, Mattera et al., 1985, Boyer et al., 1986, Sinkins and Wells, 1993, Wreggett and Wells, 1995, Chidiac et al., 1997, Jordan and Devi, 1999). Cooperativity implies oligomers, however, and early biochemical studies on muscarinic receptors suggested that multimeric forms were a minor component at best (e.g., Peterson et al., 1986, Peterson et al., 1988, Schimerlik et al., 1986, Berstein et al., 1988). In contrast, more recent data point increasingly to oligomers as a common feature of G protein-linked receptors under at least some conditions (e.g., Potter et al., 1991, Maggio et al., 1993, Wreggett and Wells, 1995, Hébert et al., 1996, Hébert et al., 1998, Monnot et al., 1996, Jordan and Devi, 1999, Zeng and Wess, 1999, Angers et al., 2000, Lee et al., 2000, Rocheville et al., 2000).

Questions regarding the role of oligomers in G protein-mediated signalling relate in part to oligomeric size and stability. Dimers and larger oligomers have been identified on Western blots of several receptors, including muscarinic receptors Wreggett and Wells, 1995, Zeng and Wess, 1999, and tetramers or larger aggregates have been inferred from functional studies Wreggett and Wells, 1995, Chidiac et al., 1997. Oligomers also have been identified by coimmunoprecipitation of differently tagged receptors, but the data presently available do not permit a distinction among different oligomeric states. Data on oligomeric stability are similarly inconclusive. Agonists have been reported to favour dimerisation of β2-adrenoceptors as revealed on Western blots (Hébert et al., 1996). Similarly, agonists and the level of expression were reported to affect the fluorescence resonance energy transfer between labelled somatostatin receptors in Chinese hamster ovary (CHO) cells (Rocheville et al., 2000). In both cases, it was suggested that agonists promote dimerisation within the membrane. In contrast, changes in bioluminescence resonance energy transfer between β-adrenoceptors have prompted the suggestion that agonist-dependent effects derive from conformational changes within the complex (Angers et al., 2000). Agonists were found to be without effect on the degree of dimerisation of muscarinic m3 receptors or κ-opioid receptors, as revealed on Western blots Jordan and Devi, 1999, Zeng and Wess, 1999.

Owing to uncertainty over the interpretation of recent data, we have used coimmunoprecipitation to investigate the size and stability of oligomers formed by c-myc- and FLAG-tagged muscarinic m2 receptors extracted from Sf9 cells. The results suggest that the receptor retains its oligomeric integrity irrespective of agonists or antagonists, and that it exists at least in part as a trimer or larger oligomer. A preliminary report of this work has appeared elsewhere (Park et al., 2001).

Section snippets

Ligands, detergents, antisera and other materials

[3H]Quinuclidinylbenzilate was obtained from NEN Life Science Products (lots 3329907 and 3363333, 42 Ci/mmol), and N-methylscopolamine bromide was from Sigma-Aldrich. Digitonin used to solubilise and purify the receptor was purchased from Wako Bioproducts at a purity near 100%. Sephadex G-50 columns used in the binding assays were pre-equilibrated and eluted with buffer solutions containing digitonin obtained from Boehringer Mannheim (purity, >75% or 93%) and Calbiochem (purity, >75%). Cholic

Results

Muscarinic m2 receptors tagged with the c-myc and FLAG epitopes were identified by their respective antibodies on Western blots prepared from singly infected and co-infected Sf9 cells. Each epitope was detected as an immunoreactive band at the position expected for the monomeric form of the receptor (i.e., 54,600±700 Da, N=13) (Fig. 1), based on the amino acid sequence (i.e., 51,715 Da). There was no reaction between the anti-c-myc antibody and the FLAG-tagged receptor or between the anti-FLAG

Discussion

Muscarinic receptors expressed in Sf9 cells form oligomers that appear to retain their multimeric status under a variety of conditions. When c-myc- and FLAG-tagged receptors were co-expressed, the two epitopes could be coimmunoprecipitated from extracts in digitonin–cholate, n-dodecyl-β-d-maltoside and Lubrol-PX. The receptors therefore exist as oligomers in the original extracts, and presumably in the cells, but their oligomeric status is unclear. On the basis of the immunoblot alone, there

Acknowledgements

This investigation was supported by the Heart and Stroke Foundation of Ontario (T2970 and T3760) and the Medical Research Council of Canada (MT14171). We are grateful to Dr. Tom Bonner of the National Institute of Mental Health for the cDNA coding for the human muscarinic m2 receptor, and to Dr. Michael Dennis, Dr. Gordon Sauvé and Normand Rondeau of Biosignal, for the baculovirus coding for the wild-type and c-myc-tagged receptors. We thankfully acknowledge Natalie Lavine for assistance in the

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