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Research ArticleCELLULAR AND MOLECULAR

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

Gregory W. Sawyer, Frederick J. Ehlert and Crystal A. Shults
Journal of Pharmacology and Experimental Therapeutics January 2010, 332 (1) 76-86; DOI: https://doi.org/10.1124/jpet.109.160986
Gregory W. Sawyer
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Frederick J. Ehlert
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Crystal A. Shults
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Abstract

We investigated the functional role of a conserved motif, F(x)6LL, in the membrane proximal C-tail of the human muscarinic M1 (hM1) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423FRDTFRLLL431. Wild-type and mutant hM1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [3H]N-methylscopolamine binding assays. The plasma membrane expression of hM1 receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM1 receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L430 and L431 play a role in folding hM1 receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V46 and L47, that, when mutated, reduce the plasma membrane expression of hM1 receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM1 receptors to achieve a transport-competent state.

Footnotes

  • This work was supported by the Oklahoma Center for the Advancement of Science and Technology [Project HR03-1072]; the Oklahoma State University, Center for Health Sciences [Grant CHS-0803]; and the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant 1R15-NS057742].

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.109.160986

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    ER
    endoplasmic reticulum
    5-HT
    5-hydroxytryptamine
    PCR
    polymerase chain reaction
    GFP, ANOVA
    analysis of variance
    GFP
    green fluorescent protein
    EGFP
    enhanced green fluorescence protein
    hM1
    human muscarinic M1 acetylcholine receptor
    CHO
    Chinese hamster ovary
    PBS
    phosphate-buffered saline
    [3H]NMS
    [3H]N-methylscopolamine
    TM1
    transmembrane spanning domain 1
    [3H]QNB
    [3H]3-quinuclidinyl benzilate
    SR121463B
    satavapatan.

    • Received August 25, 2009.
    • Accepted October 19, 2009.
  • © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 332 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 332, Issue 1
1 Jan 2010
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Research ArticleCELLULAR AND MOLECULAR

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

Gregory W. Sawyer, Frederick J. Ehlert and Crystal A. Shults
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 76-86; DOI: https://doi.org/10.1124/jpet.109.160986

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Research ArticleCELLULAR AND MOLECULAR

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

Gregory W. Sawyer, Frederick J. Ehlert and Crystal A. Shults
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 76-86; DOI: https://doi.org/10.1124/jpet.109.160986
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