Contrasting Effects of Allosteric and Orthosteric Agonists on M₁Muscarinic Acetylcholine Receptor Internalization and Down-regulation

Abstract

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allostericagonists on M 1 mACh receptor internalization and downregulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M 1 mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M 1 mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M 1 cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M 1 mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression. proteins and inhibition of adenylyl cyclase activity (Caulfield and Birdsall, 1998). The mACh receptors are widely distributed throughout the periphery and central nervous system, and have been implicated as potential therapeutic targets in an array of diseases, including chronic obstructive pulmonary disease, irritable bowel syndrome, Alzheimer's disease, and schizophrenia (see Eglen et al., 1999;Felder et al., 2000;Langmead et al., 2008b).A major obstacle in the development of successful cholinergic therapies has been the lack of mACh receptor subtypespecific ligands, and the potentially serious side-effect profile of agents that interact with multiple mACh receptor subtypes (Bymaster et al., 1998;Eglen et al., 1999;Bartolomeo

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

“…Interestingly, unlike CP55940-induced ERK1/2 phosphorylation, ORG27569-induced ERK1/2 phosphorylation is G i protein-independent. Thus, it possesses some agonistic properties as an "ago-allosteric modulator" (33)(34)(35)(36)(37)(38).…”

Allosteric Modulator ORG27569 Induces CB1 Cannabinoid Receptor High Affinity Agonist Binding State, Receptor Internalization, and Gi Protein-independent ERK1/2 Kinase Activation

“…More recently, it has become evident that some allosteric modulators can also activate the receptor for signal transduction on their own (termed "allosteric agonist") (63,64). In addition to modulating the binding and efficacy of an orthosteric ligand, these allosteric agonists were shown to directly affect receptor activation, cellular localization, or down-regulation of various GPCRs including the M1 muscarinic acetylcholine receptor, mGluR7, and adenosine A1 receptor (65)(66)(67)(68). Here, we show that ORG27569 can activate CB1 and promote downstream effects in the absence of the orthosteric agonist.…”

The muscarinic system, cognition and schizophrenia

“…Drugs have typically been developed to target the orthosteric binding site for the endogenous ligands of G protein-coupled receptors, however the five muscarinic receptors share considerable orthosteric binding site homogeneity (see Eglen, 2005Eglen, , 2006. As a consequence it is very difficult to target a specific muscarinic receptor at this site without manipulating any of the remaining four receptors and producing unwanted side-effects and/or receptor down-regulation/drug desensitization (Lenz et al, 1994;Mirza et al, 2003;Mullaney et al, 1993;Thomas et al, 2009). Fortunately, it has been discovered that the M1 (Espinoza-Fonseca and Trujillo-Ferrara, 2005), M2 (Holzgrabe et al, 2000), M3 (Jooste et al, 2005), M4 (Kennedy et al, 2009) and M5 receptors all exhibit a secondary, allosteric binding site.…”