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Molecular and Cellular Biology, March 2004, p. 2041-2051, Vol. 24, No. 5
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.5.2041-2051.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled -Factor Receptor

Jennifer C. Lin,¹ Ken Duell,² and James B. Konopka^2*

Graduate Program in Molecular and Cellular Biology,¹ Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222²

Received 9 July 2003/ Returned for modification 7 October 2003/ Accepted 21 November 2003

The -factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

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* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794-5222. Phone: (631) 632-8715. Fax: (631) 632-9797. E-mail: james.konopka{at}sunysb.edu.

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Molecular and Cellular Biology, March 2004, p. 2041-2051, Vol. 24, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.5.2041-2051.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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• Lee, Y.-H., Naider, F., Becker, J. M. (2006). Interacting Residues in an Activated State of a G Protein-coupled Receptor. J. Biol. Chem. 281: 2263-2272 [Abstract] [Full Text]