Co-operative and oligomeric potential of the M2 muscarinic cholinergic receptor.
Published: 2004
Language: eng
Number of pages: 286
Description: G protein-mediated signalling has been suggested to occur via co-operative interactions within an oligomeric receptor. In the current study, non-competitive effects have been observed in the binding of the muscarinic antagonists  N-methylscopolamine (NMS) and quinuclidinylbenzilate (QNB) to M 2 muscarinic receptors extracted from porcine atria in cholate-NaCl or Lubrol-PX. In particular, the apparent capacity for [3H]QNB exceeds that for [3H]NMS, but unlabelled NMS is inhibitory at all of the sites labelled by [3H]QNB. NMS therefore appears to inhibit at sites to which it does not bind. The data can be described in terms of co-operative interactions within a receptor that is tetravalent or larger and retains its oligomeric stability under the conditions of the assays. Similar effects also have been observed with the M2 muscarinic receptor from baculoviral infected Sf9 cells. In that case, however, the manifestation of cooperativity was dependent upon the level of cholesterol in the membrane; moreover, at least eight interacting sites were required to describe the data.Direct evidence for oligomers has come from the co-immunoprecipitation of c-Myc- and FLAG-tagged M2 muscarinic receptors in various detergent-solubilised extracts of co-infected Sf9 cells. In digitonin-cholate, the receptor appears to exist as a static mixture of monomers and oligomers. The two forms of the receptor have been separated on the basis of binding activity and immunological reactivity. Monomers appear to be functional insofar as they bind antagonists with typical musarinic affinity, thereby raising the possibility that they also participate in the signalling process. The oligomers are wholly or largely inactive, but their function can be preserved or recovered by treatment of membranes with an antagonist prior to solubilisation. The possibility that the M2 muscarinic receptor can form relatively large oligomers is supported by the detection of a heteromer containing c-Myc-, FLAG-, and HA-tagged forms of the receptor in extracts of triply infected Sf9 cells. M2 muscarinic receptors therefore exhibit co-operativity in the binding of ligands to interacting sites within an oligomer that is tetrameric and very likely larger. These observations are consistent with the notion that co-operative interactions lie in the mechanistic pathway of signalling via G protein-coupled receptors.
Notes: Adviser:  James W. Wells.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2427.