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Abstract
Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.
Highlights
The 5 subtypes of muscarinic acetylcholine receptors3 exemplify the group of G protein-coupled receptors (GPCRs) activated by the reversible binding of small ligands [1]. mAChRs are known classically to be the mediators of the parasympathetic actions of acetylcholine (ACh), but recent gene knock-out studies have emphasized their importance in modulating synaptic plasticity in the central nervous system [2]
In the M1 mAChR, Asn substitution first established the critical role of Asp71 in signal transduction [13], a finding subsequently confirmed in the M2 [14] and M3 [15] subtypes as well as a host of other receptor types [16]
The aim of this study was to make a systematic analysis of the functional roles of all of the amino acid side chains from positions 2.40 (Asn61) to 2.60 (Leu81) of the M1 mAChR by alanine substitution mutagenesis (Ala itself was mutated to Gly)
Summary
The 5 subtypes of muscarinic acetylcholine receptors (mAChRs) exemplify the group of G protein-coupled receptors (GPCRs) activated by the reversible binding of small ligands [1]. mAChRs are known classically to be the mediators of the parasympathetic actions of acetylcholine (ACh), but recent gene knock-out studies have emphasized their importance in modulating synaptic plasticity in the central nervous system [2]. NOVEMBER 2, 2007 VOLUME 282 NUMBER 44 of the ACh-induced phosphoinositide (PI) turnover and extracellular signal-regulated kinase (ERK) kinase activation [3], and help to mediate the enhancement of hippocampal long-term potentiation by physiologically released ACh [4] The importance of these processes in cognition and memory motivates efforts to understand the structure and activation of M1 mAChRs, especially for the purposes of selective drug development [5]. The most systematic studies have been a substituted cysteine scan from residues 2.47 to 2.68 in the D2 receptor [23], and random mutagenesis from residues 2.43 to 2.63 of the C5a receptor [24] In the former case, the accessibility of the mutant side chains to sulfhydryl reagents was the issue, whereas in the latter the criterion was preservation of receptor signaling, which defined important positions but did not directly address the function of the side chains. This study confirmed the functional importance of the highly conserved amino acids
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