The M ₃ -muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner

Abstract

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M 1 -muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M 3 -muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M 3 -muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M 3 -muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M 3 -muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M 3 -muscarinic receptor was coupled normally to G q/11 -signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M 3 -muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M 3 -muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.A mong the multitude of physiological responses regulated by G protein-coupled receptors (GPCRs), one of the most intriguing is the ability of this superfamily of cell-surface receptors to regulate neurological and behavioral processes such as learning and memory (1-4). The members of the muscarinic acetylcholine receptor family are prominent among the GPCR subtypes associated with cognitive function because lesions in cholinergic innervations to the hippocampus and other brain areas are widely thought to underlie the cognitive deficit observed in Alzheimer's disease (5). Whereas the M 1 -muscarinic receptor subtype has been proposed to be the subtype associated with acetylcholine-mediated cognition (6, 7), recent gene-knockout experiments have cast doubt on the direct role of this receptor subtype in learning and memory (1,8). This has been reinforced by the discovery of a novel selective M 1 -muscarinic receptor antagonist that was effective in blocking M 1 -muscarinic receptor-mediated seizures in vivo but had no effect on hippocampal-based contextual fear conditioning (9). In addition, recent studies using an M 1 -muscarinic receptorpositive allosteric modulator, BQCA, have suggested that M 1 -muscarinic receptors can mediate learning and memory through an indirect mechanism by stimulating the prefrontal cortex (10). There is some con...

M 3 -muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1

“…Our recent studies established that removal of the phosphorylation sites on the M 3 -muscarinic receptor resulted in a deficit in the recruitment of β-arrestin (14). This result was confirmed here using the PathHunter β-arrestin recruitment assay and the human M 3 -muscarinic receptor containing comparable phosphoacceptor site mutations to that of the mouse receptor.…”

“…To test the physiological role of M 3 -muscarinic receptor phosphorylation, we generated a knock-in mouse strain via homologous recombination that replaced the coding sequence of the wild-type M 3 -muscarinic receptor with that of the phosphorylation-deficient, M3 phos-neg receptor (14). We have previously reported that homozygous mice for this gene-targeting event (designated M3R-KI) displayed no gross physiological defects and showed normal Mendelian breeding (14). Furthermore, the weight and food consumption of the M3R-KI mice was not significantly different from wild-type animals (Fig.…”

“…To define the potential physiological role of M 3 -muscarinic receptor phosphorylation, we generated a mouse strain where the wild-type M 3 -muscarinic receptor gene had been replaced by a phosphorylationdeficient mutant form of the receptor. Using this mouse model, we have recently demonstrated an important role for receptor phosphorylation and β-arrestin signaling in muscarinic receptor-mediated hippocampal memory and learning (14). In the present study we focus on the role of M 3 -muscarinic receptor phosphorylation in the regulation of glucose-dependent insulin release from pancreatic islets, a response that is known to be augmented by parasympathetic pathways acting on M 3 -muscarinic receptors expressed on pancreatic β-cells (15-17).…”

Muscarinic acetylcholine receptors act in synergy to facilitate learning and memory

“…This is supported by work showing that both M 3 knockout mice and mice with M 3 phosphorylation deficiency have deficits in contextual fear conditioning (Poulin et al 2010). Interestingly, whereas M 1 rather than M 3 is the predominant mediator of muscarinic potentiation of hippocampal LTP (Anagnostaras et al 2003;Shinoe et al 2005;Anisuzzaman et al 2013;Dennis et al 2016), a putative physical substrate for learning, M 3 modulates the inhibition of excitatory synaptic transmission in CA1 (de Vin et al 2015).…”

“…Although pharmacological and electrophysiological approaches often point to M 1 or M 2 as potent mediators of learning (Sen and Bhattacharya 1991;Fornari et al 2000;Power et al 2003;Soares et al 2006;Figueredo et al 2008;Ma et al 2009), constitutive knockout of these receptors has no effect on learning in a variety of tasks (Anagnostaras et al 2003;Bainbridge et al 2008). Moreover, when such manipulations do impact learning, as is the case with constitutive M 3 deletion (Poulin et al 2010), the neuroanatomical basis for the effect and the specific memory process(es) affected remain unknown.…”