Constitutive Activity and Inverse Agonism at the M₂Muscarinic Acetylcholine Receptor

Abstract

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M 2 muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [ 3 H]inositol phosphates in cells coexpressing the chimeric G␣ qi5 protein and the N410Y mutant M 2 mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M 2 mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M 2 mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [ 3 H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of G␣ i/o versus G␣ s , consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M 2 mACh receptor. This indicates that the N410Y mutation of the M 2 mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.A crucial development in our understanding of G-proteincoupled receptor (GPCR) function has been the identification of the ability of receptors to activate their cognate G-proteins in the absence of an agonist (Costa and Herz, 1989). Thus, certain ligands (termed inverse agonists and previously characterized as competitive antagonists) can inhibit agonist-independent receptor activity (Costa and Herz, 1989). Subsequent research has identified significant agonist-independent (constitutive) activity at a wide variety of both endogenously and recombinantly expressed GPCRs (for review, see Seifert and Wenzel-Seifert, 2002).One of the most powerful tools used by researchers in this area has been the development of GPCRs harboring specific mutations known to enhance the agonist-independent coupling of receptor and G-protein [so-called constitutively active mutant (CAM) receptors] (Seifert and Wenzel-Seifert, 2002). Mutations in a number of well conserved domains, including the D/ERY motif at the intracellular interface of the third transmembrane domain (TM3) and the BBXXB motif (where B is Arg or Lys) toward the C-terminal end of the third intracellular loop, have been reported to ...

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1Acetylcholine Receptors Affects Plasma Membrane Expression

“…The similarity between the two estimates shows that the atropine-rescued hM 1AA430 -431 receptor exhibits stability comparable with that of the wild-type hM 1 receptor, and that the effect of atropine cannot be attributed to prevention of receptor internalization and degradation. The hM 1AA430 -431 mutant behaves differently from M 2 and M 3 muscarinic receptors bearing a N6.58Y mutation that causes internalization and constitutive activation in an atropinereversible manner (Dowling et al, 2006;Nelson et al, 2006).…”

Comparison of the Activation Kinetics of the M3Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

“…As outlined in the Introduction, the position corresponding to amino acid 514 in the human M 3 -AChR was shown to generate constitutive activity in all muscarinic receptor subtypes (Dowling et al, 2006;Nelson et al, 2006). In particular, the mutation N514Y was reported to cause constitutive activity in the M 3 -AChR and was analyzed in detail (Dowling et al, 2006).…”

“…Several different mutations have been demonstrated to cause constitutive receptor activity for the muscarinic acetylcholine receptor family (Spalding and Burstein, 2006). In particular, the amino acid in position 6.58 [according to the Ballesteros/ Weinstein numbering scheme (Ballesteros and Weinstein, 1995)] at the interface of transmembrane domain (TM) 6 and the extracellular loop was shown to cause constitutive activity in all five muscarinic AChR subtypes (Spalding et al, 1997;Ford et al, 2002;Dowling et al, 2006;Nelson et al, 2006), whereas alternative mutations in the E/DRY motif in TM3 or in the NPXXY motif in TM7 close to the cytoplasmic side (Spalding and Burstein, 2006;Smit et al, 2007) did not cause constitutive activity at the M 1 -AChR. This particular position corresponds to amino acid 514 in the human M 3 -AChR, and the mutation N514Y within the human M 3 -AChR has been thoroughly characterized for its effects on basal receptor activity, agonist affinity, and inverse agonist effects (Dowling et al, 2006).…”

Mechanisms of M3 Muscarinic Receptor Regulation by Wash-Resistant Xanomeline Binding

“…These discrepancies may be due to unique interactions of atropine with the receptor. While commonly described as an antagonist, atropine also exhibits properties of an inverse agonist [39,40] . For example, previous research found that continuous treatment with atropine can lead to an increase in receptor expression [23,29] .…”