Evaluation of a Chemical Genetic Approach to Determine GPCR Function.

SM Brooke, AB Tobin. MRC Toxicology Unit, Leicester, UK

The M1 muscarinic acetylcholine (ACh) receptor is a member of a family of 5 GPCRs, termed M1-M5, found within the central and peripheral nervous systems. The M1 muscarinic receptor is also found at high levels in regions such as the hippocampus, cerebral cortex, and the striatum (1). Their presence in these regions of the brain, linked with learning and memory, has led to research into the receptors involvement in neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, as well as in neurological conditions such as schizophrenia.

Through the development of mutant receptors it is possible to investigate the functions of receptors in vitro and in vivo, as well as the downstream signalling effectors of different signalling pathways. Receptors activated solely by synthetic ligands (RASSLs), also known as designer receptors exclusively activated by synthetic ligands (DREADDs), have had their binding pocket modified so that it no longer binds to the natural ligand, but will bind to an inert synthetic ligand. As they will no longer interact with their natural ligand it is possible to use these receptors to knock-out receptor function and instead knock-in receptor function by administering the synthetic ligand. This dynamic ability to knock-out and knock-in receptor function allows for more direct observations as to the functions of the receptor. Once the receptors are shown to activate the same downstream effectors as the wild type receptor it is possible to use these receptors for the determination of GPCR function; as well as in in vitro and in vivo drug discovery roles. The overall aims of this project are to understand the role the M1 muscarinic receptor plays in neurodegenerative disorders, investigating its potential as a therapeutic target, and assessing the usefulness of a chemical genetic approach in the determination of GPCR Function. As can be seen in Table 1, the mutations in the RASSL receptor have removed the ability of ACh to activate the receptor except for at maximal concentration. Clozapine-N-oxide (CNO), an agonist for the RASSL receptor, only acts as a partial agonist towards the wild type receptor at maximal concentrations.

Table 1: Concentration dependent increases in ERK activation in M1-WT HA CHO FLPIn and M1-RASSL HA CHO FLPIn cell lines treated with ACh and CNO. Magnitude of response measured as fold over basal. Data are shown as mean ± SEM. For pEC50 (M) values; n=3

There is a large change in the EC50 of ACh between the WT and RASSL receptors shows that the mutations have effectively silenced the signalling by ACh through the M1 receptor. This silencing supports the potential of the RASSL receptor as an in vivo disease model for the loss of M1 function. The partial agonism of CNO against the WT receptor is at low levels in maximal concentrations of CNO but need to be investigated with the other members of the muscarinic family; signalling initiated by CNO though other members of the family could complicate interpretation of results.

(1) Abrams, P. et al, BJP, 5:148 (2006)