Hulme group ::

Muscarinic acetylcholine receptors

The seven-transmembrane domain ( 7-TM) receptors are the largest family of cell-surface signalling molecules in the human genome. The muscarinic acetylcholine receptors are important members of the 7-TM superfamily. There are five mAChR subtypes, each with a unique distribution. Signalling through the mAChRs is mediated by direct activation of heterotrimeric GTP-binding proteins (G-proteins), with the additional involvement of small G-proteins, and tyrosine kinases. Muscarinic signals activate and inhibit neurons in the central and peripheral nervous systems, induce secretion from gland cells, and regulate the contraction of cardiac and smooth muscle. They can cause long-term modulation of synaptic efficacy, which is necessary for memory. Thus, mAChRs are important pharmacological targets. Investigation of the mAChRs is important for understanding the role of cholinergic synapses in the brain. Further, it provides a paradigm for understanding more general aspects of GPCR signalling.

We wish to understand the activation of mAChRs in molecular detail. One approach is to use mutations to study receptor function. Using scanning and point mutagenesis, we have located many of the amino acids that bind ACh, and the receptor’s partner GTP-binding protein. By combinatorial mutagenesis techniques, we have found a network of conserved intramolecular contacts that stabilise the three-dimensional structure of the receptor. These contacts help to constrain the receptor to the inactive state in the absence of the ACh. Their rearrangement is necessary for the conformational switch from the inactive to the active state of the receptor. The findings are being fitted into increasingly refined models of the structure of the receptor. Another strategy is to determine the three-dimensional structure of the mAChRs at atomic resolution using X-ray crystallography. We produce milligramme quantities of purified mAChRs using over-expression systems in combination with engineering of the receptor sequence to stabilise the structure, and to aid purification. Crystallographic studies are underway.

7-TM receptor signalling is highly divergent, and can transactivate novel signalling pathways, mediated by tyrosine kinases, small G-proteins, and membrane-bound metalloproteases. Phosphorylation of signalling molecules, juxtacrine signalling, and gene transcription induced by the activation of kinase cascades, such as the MAP kinase pathway, may underlie the ability of muscarinic receptors to modulate long-term synaptic potentiation in the brain. We are attempting to dissect and reconstitute these signalling pathways in cell lines in vitro so that we can understand and manipulate the interactions of the various components. We are applying the lessons learned to neurons.

A model of the muscarinic receptor ::

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A model of the muscarinic receptor showing (A) an antagonist and (B) acetylcholine docked into the binding site.

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