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PMID: 21247934 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Helix 8 of the M1 muscarinic acetylcholine receptor: scanning mutagenesis delineates a G protein recognition site.

Molecular pharmacology ·Vol. 79 ·No. 4 ·2011-04-00 ·Pages 701-9

Kaye RG, Saldanha JW, Lu ZL, Hulme EC

Abstract

We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M(1) muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein α subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.

MeSH Terms
Alanine/genetics Animals Binding Sites/genetics COS Cells Chlorocebus aethiops Dose-Response Relationship, Drug GTP-Binding Proteins/chemistry,genetics,metabolism Mutagenesis/genetics Protein Structure, Secondary/genetics Protein Structure, Tertiary/genetics Rats Receptor, Muscarinic M1/chemistry,genetics,metabolism
Chemicals
Receptor, Muscarinic M1 GTP-Binding Proteins Alanine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Kaye Robert G
Division of Physical Biochemistry, MRC National Institute for Medical Research, London, United Kingdom.
Saldanha José W
Lu Zhi-Liang
Hulme Edward C
References (40)
40 references, click to expand
  1. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor.
    J Biol Chem. 1989 May 5;264(13):7564-9 PMID: 2540197
  2. Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism.
    J Biol Chem. 2006 Feb 17;281(7):4109-16 PMID: 16354660
  3. Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands.
    J Biol Chem. 2007 Sep 7;282(36):26284-93 PMID: 17623649
  4. Structural basis of G protein-coupled receptor-G protein interactions.
    Nat Chem Biol. 2010 Jul;6(7):541-8 PMID: 20512139
  5. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.
    Science. 2008 Nov 21;322(5905):1211-7 PMID: 18832607
  6. The dynamic role of palmitoylation in signal transduction.
    Trends Biochem Sci. 1995 May;20(5):181-7 PMID: 7610481
  7. Crystal structure of rhodopsin: A G protein-coupled receptor.
    Science. 2000 Aug 4;289(5480):739-45 PMID: 10926528
  8. Structure of a beta1-adrenergic G-protein-coupled receptor.
    Nature. 2008 Jul 24;454(7203):486-91 PMID: 18594507
  9. Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.
    Biochemistry. 1999 Jun 22;38(25):7925-30 PMID: 10387034
  10. A conserved motif in the membrane proximal C-terminal tail of human muscarinic m1 acetylcholine receptors affects plasma membrane expression.
    J Pharmacol Exp Ther. 2010 Jan;332(1):76-86 PMID: 19841475
  11. The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis.
    J Biol Chem. 1999 Mar 12;274(11):7309-15 PMID: 10066794
  12. Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study.
    Biochemistry. 1999 Jun 22;38(25):7931-7 PMID: 10387035
  13. Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation.
    Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2290-5 PMID: 12601165
  14. Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands.
    Mol Pharmacol. 2007 Dec;72(6):1484-96 PMID: 17848601
  15. Helix 8 of the leukotriene B4 receptor is required for the conformational change to the low affinity state after G-protein activation.
    J Biol Chem. 2003 Oct 17;278(42):41500-9 PMID: 12902330
  16. Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor.
    J Biol Chem. 2007 Nov 2;282(44):32471-9 PMID: 17823120
  17. Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy.
    Biochemistry. 2008 Nov 4;47(44):11518-27 PMID: 18847221
  18. Site-directed mutagenesis of the rat m1 muscarinic acetylcholine receptor. Role of conserved cysteines in receptor function.
    J Biol Chem. 1992 Jun 5;267(16):11439-48 PMID: 1317867
  19. Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.
    EMBO J. 2009 Nov 4;28(21):3315-28 PMID: 19763081
  20. Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors.
    Biochem Soc Trans. 2007 Aug;35(Pt 4):742-5 PMID: 17635138
  21. Three cytoplasmic loops of rhodopsin interact with transducin.
    Proc Natl Acad Sci U S A. 1989 Sep;86(18):6878-82 PMID: 2780545
  22. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.
    Science. 2010 Nov 19;330(6007):1066-71 PMID: 20929726
  23. Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.
    Biochemistry. 1993 Nov 16;32(45):12025-32 PMID: 8218279
  24. A domain for G protein coupling in carboxyl-terminal tail of rat angiotensin II receptor type 1A.
    J Biol Chem. 1997 Sep 19;272(38):23631-6 PMID: 9295303
  25. Crystal structure of squid rhodopsin.
    Nature. 2008 May 15;453(7193):363-7 PMID: 18480818
  26. Mutagenic mapping suggests a novel binding mode for selective agonists of M1 muscarinic acetylcholine receptors.
    Mol Pharmacol. 2009 Feb;75(2):331-41 PMID: 19001633
  27. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.
    Science. 2007 Nov 23;318(5854):1258-65 PMID: 17962520
  28. Mapping of single amino acid residues required for selective activation of Gq/11 by the m3 muscarinic acetylcholine receptor.
    J Biol Chem. 1995 Jul 28;270(30):17741-8 PMID: 7629074
  29. Characterization of the residues in helix 8 of the human beta1-adrenergic receptor that are involved in coupling the receptor to G proteins.
    J Biol Chem. 2006 May 5;281(18):12896-907 PMID: 16500896
  30. The function of a highly-conserved arginine residue in activation of the muscarinic M1 receptor.
    Eur J Pharmacol. 1995 Feb 15;288(3):251-7 PMID: 7774669
  31. Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms.
    J Biol Chem. 2007 Aug 24;282(34):25100-13 PMID: 17595161
  32. A G protein-coupled receptor at work: the rhodopsin model.
    Trends Biochem Sci. 2009 Nov;34(11):540-52 PMID: 19836958
  33. Seven-transmembrane receptors: crystals clarify.
    Trends Pharmacol Sci. 2002 Mar;23(3):140-6 PMID: 11879682
  34. The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis.
    Eur J Pharmacol. 1995 May 26;289(3):429-37 PMID: 7556411
  35. Structure of bovine rhodopsin in a trigonal crystal form.
    J Mol Biol. 2004 Nov 5;343(5):1409-38 PMID: 15491621
  36. Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations.
    J Biol Chem. 2002 Sep 27;277(39):36577-84 PMID: 12145300
  37. Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.
    J Biol Chem. 2001 Sep 7;276(36):34098-104 PMID: 11441014
  38. Muscarinic receptor subtypes.
    Annu Rev Pharmacol Toxicol. 1990;30:633-73 PMID: 2188581
  39. Crystal structure of opsin in its G-protein-interacting conformation.
    Nature. 2008 Sep 25;455(7212):497-502 PMID: 18818650
  40. Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated.
    FEBS Lett. 1988 Mar 28;230(1-2):1-5 PMID: 3350146
Article Info
Journal
Molecular pharmacology
Abbr.
Mol Pharmacol
ISSN
1521-0111
DOI
10.1124/mol.110.070177
Published
2011-04-00
Epub
2011-00-19
Pages
701-9
Language
English
Region
United States
NLM ID
0035623
PMCID
PMC3063726
Subset
IM
Grants
Medical Research Council · MC_U117532184 · United Kingdom
Medical Research Council · MC_U127685846 · United Kingdom
Medical Research Council · U117532184 · United Kingdom
Medical Research Council · U117581331 · United Kingdom
External Links
PubMed source DOI: 10.1124/mol.110.070177 PMC Full Text View journal details
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