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Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors

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Purpose

This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1–M5), a region important for G protein coupling.

Methods

GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues.

Results

28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, Δ232–358.

Conclusion

Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.

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Abbreviations

CAm:

calmodulin

GPCR:

G-protein coupled receptor

hM1–3:

human muscarinic acetylcholine receptor, types 1–3

mAchR:

muscarinic acetylcholine receptor

mGluR:

metabotropic glutamate receptor

MOR:

μ opioid receptor

PKC:

protein kinase C

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Acknowledgment

This work was supported by NIH Research Grant DA04166, from the National Institute on Drug Abuse.

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Author notes

  1. Julie L. Lucas

    Present address: Berlex Biosciences, 2600 Hilltop Drive, P.O. Box 4099, Richmond, California, 94804-0099, USA

Authors and Affiliations

  1. Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, California, 94143-0446, USA

    Julie L. Lucas

  2. Department of Pharmacology, College of Medicine and Public Health, Ohio State University, Columbus, Ohio, 43221-1239, USA

    Julie L. Lucas, Danxin Wang & Wolfgang Sadée

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  1. Julie L. Lucas
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Correspondence to Julie L. Lucas.

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Lucas, J.L., Wang, D. & Sadée, W. Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors. Pharm Res 23, 647–653 (2006). https://doi.org/10.1007/s11095-006-9784-9

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  • DOI: https://doi.org/10.1007/s11095-006-9784-9

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