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Rapid identification of functionally critical amino acids in a G protein–coupled receptor

Nature Methods volume 4, pages 169–174 (2007)Cite this article

Abstract

G protein–coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.

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Figure 1: Summary of the selection strategy used to identify point mutations that abolish M3R function in yeast.
Figure 2: Characterization of M3R expression and signaling in yeast.
Figure 3: Summary of point mutations that abolish or greatly impair M3R function in mammalian (COS-7) cells.
Figure 4: Predicted location of functionally important TM I and II residues in the three-dimensional structure of the M3R.

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Acknowledgements

This article is dedicated to the memory of Bo Li who died on October 20, 2006, in a tragic traffic accident. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Disorders (NIDDK). We thank Dr. P. William (NIH-NIDDK) for his help with the microscopic studies.

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Author notes

  1. Bo Li: Deceased.

Authors and Affiliations

  1. Molecular Signaling, Bethesda, 20892, Maryland, USA

    Bo Li, Marco Scarselli, Christopher D Knudsen, Sara M McMillin & Jürgen Wess

  2. Molecular Recognition Sections, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, 20892, Maryland, USA

    Soo-Kyung Kim & Kenneth A Jacobson

Authors
  1. Bo Li
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Correspondence to Jürgen Wess.

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Supplementary information

Supplementary Table 1

A C-terminal EGFP tag has no significant effect on the ligand binding and functional properties of the full-length rat M3R transiently expressed in COS-7 cells.

Supplementary Table 2

Use of error-prone PCR to generate yeast libraries expressing randomly mutagenized mutant M3Rs.

Supplementary Table 3

Complexity of the yeast libraries expressing randomly mutagenized mutant M3Rs generated via error-prone PCR.

Supplementary Table 4

Characterization of mutant M3Rs recovered in the yeast genetic screen in transfected COS-7 cells.

Supplementary Note 1

Functional activity in yeast of mutant M3Rs containing amino acid substitutions at position Leu225.

Supplementary Note 2

Mutant M3Rs displaying reduced Bmax values and cell surface expression.

Supplementary Note 3

Predicted location and interactions of functionally important TM I and II residues in the 3D structure of the M3R.

Supplementary Methods

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Li, B., Scarselli, M., Knudsen, C. et al. Rapid identification of functionally critical amino acids in a G protein–coupled receptor. Nat Methods 4, 169–174 (2007). https://doi.org/10.1038/nmeth990

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