All five subtypes of muscarinic acetylcholine receptor (M1-M5) are expressed in the CNS and they are implicated in such processes as learning, memory, and locomotion. Alterations in central cholinergic activity are thought to contribute to the pathophysiology of certain neurological disorders, including Alzheimer's disease and schizophrenia. Current efforts to develop subtype-selective muscarinic agonists (i.e., M1-selective agonists) to treat these and other neurological disorders are based on the anticipated functional roles of the receptors in the CNS. To date, the effects of agonist on the plasma membrane expression and second messenger signaling of muscarinic receptors expressed in the CNS are poorly described yet, knowledge of these fundamental responses to agonist exposure is necessary for the development of efficacious therapeutics with minimal side effects. Consequently, the long-term goal of this application is to develop an understanding of the molecular mechanisms mediating agonist-induced internalization of subtypes of muscarinic receptor expressed in the CNS and the effect of this process on receptor signaling. Consistent with this goal, the primary objective of this AREA application is to characterize the effect of agonist exposure on muscarinic receptor plasma membrane expression in cortical neurons and to determine the role a small M1 receptor domain plays in this process. To accomplish the objective of this application, the following hypotheses will be tested: 1. Muscarinic receptors expressed in primary cortical neurons will rapidly and extensively internalize when exposed to muscarinic agonist (Specific Aim 3). 2. Deletion of and/or point mutations at critical positions within a small domain in the third intracellular (i3) loop of M1 receptors will inhibit agonist-induced internalization in cortical neurons without effecting ligand affinity or agonist potency (Specific Aims 1 and 3). 3. Cys residues within this small i3 loop domain mediate M1 receptor dimerization (Specific Aims 2). The primary experimental approach devised to test these hypotheses is to express wild-type and mutant M1 receptors in CHO cells and in primary cortical neurons prepared from M1 and M4 receptor double knockout mice, and then characterize the kinetics and extent of internalization to agonist using a pharmacological approach. Collectively, this investigation is expected to substantially add to the current knowledge of muscarinic receptor trafficking in neurons of the CNS and to identify the molecular determinants of the receptor necessary for agonist-induced internalization to occur. The Public Health Relevance: The primary goal of the research proposed in this application is to further characterize the role a small domain plays in the agonist-induced internalization of M1 receptors and to characterize the kinetics of muscarinic receptor internalization in primary cortical neurons. The relevance of the proposed project to public health is that it will add to our current knowledge of the effect of agonist on muscarinic receptor plasma membrane expression in neurons of the CNS. Ultimately, this knowledge may lead to an understanding of the underlying mechanism and the identification of new therapeutic targets that can be used to alter agonist-dependent changes in muscarinic receptor plasma membrane expression. ? ? ?
The primary goal of the research proposed in this application is to further characterize the role a small domain plays in the agonist-induced internalization of M1 receptors and to characterize the kinetics of muscarinic receptor internalization in primary cortical neurons. The relevance of the proposed project to public health is that it will add to our current knowledge of the effect of agonist on muscarinic receptor plasma membrane expression in neurons of the CNS. Ultimately, this knowledge may lead to an understanding of the underlying mechanism and the identification of new therapeutic targets that can be used to alter agonist-dependent changes in muscarinic receptor plasma membrane expression.
| Thangaraju, Arunkumar; Sawyer, Gregory W (2011) Comparison of the kinetics and extent of muscarinic M1-M5 receptor internalization, recycling and downregulation in Chinese hamster ovary cells. Eur J Pharmacol 650:534-43 |
| Sawyer, Gregory W; Ehlert, Frederick J; Shults, Crystal A (2010) A conserved motif in the membrane proximal C-terminal tail of human muscarinic m1 acetylcholine receptors affects plasma membrane expression. J Pharmacol Exp Ther 332:76-86 |
| Suga, Hinako; Sawyer, Gregory W; Ehlert, Frederick J (2010) Mutagenesis of nucleophilic residues near the orthosteric binding pocket of M1 and M2 muscarinic receptors: effect on the binding of nitrogen mustard analogs of acetylcholine and McN-A-343. Mol Pharmacol 78:745-55 |
