Investigating Allosteric Activation of the M1 Muscarinic Acetylcholine Receptor
thesisposted on 06.05.2011, 11:16 by Rachel Thomas
Allosteric ligands of G protein-coupled receptors (GPCRs) bind to sites that are topographically distinct from the orthosteric site. AC-42 and 77-LH-28-1 are functionally selective M1 muscarinic acetylcholine (mACh) receptor allosteric agonists that are able to activate the M1 mACh receptor in the absence of an orthosteric ligand. In the present study, a variety of signalling pathways activated by AC-42 and 77-LH-28-1 have been investigated and compared with those activated by orthosteric agonists in Chinese hamster ovary (CHO) cells recombinantly expressing human M1 mACh receptors. Both orthosteric and allosteric agonists are able to activate Gαq/11-dependent signalling as demonstrated by concentration-dependent increases in [35S]-GTPγS binding to Gαq/11 subunits, [³H]-inositol phosphate accumulation and Ca²+ mobilisation. Both AC-42 and 77-LH-28-1 are also able to activate extracellular signal-regulated kinase 1/2 and cyclic AMP response-element binding protein (CREB). However, while all agonists enhance forskolin-stimulated cyclic AMP accumulation, only orthosteric agonists cause significant increases in [35S]-GTPγS binding to Gαi-proteins, suggesting that subtle differences may exist in the receptor conformations stabilised by orthosteric versus allosteric ligands. The effects of orthosteric and allosteric agonists on the regulation of the M1 mACh receptor expressed in CHO cells revealed that in contrast to orthosteric agonists, which cause significant internalisation and down-regulation, prolonged exposure to AC-42 does not significantly alter either cell-surface or total cellular M1 mACh receptor expression. 77-LH-28-1 does cause receptor internalisation, but not down-regulation. The apparent inability of AC-42 to cause M1 mACh receptor desensitisation is supported by the observation that arecoline was still able to stimulate a similar phosphoinositide hydrolysis response in CHO-hM1 cells incubated for 24 h with AC-42. These data indicate that AC -42 binding causes functional signalling in the absence of receptor regulatory mechanisms. These distinct pharmacological properties of allosteric agonists may provide therapeutic advantages additional to receptor subtype selectivity of action.