HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 4, 2263-2272, January 27, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/4/2263    most recent M509987200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, Y.-H. Articles by Becker, J. M. Search for Related Content PubMed PubMed Citation Articles by Lee, Y.-H. Articles by Becker, J. M. Social Bookmarking                      What's this?

Interacting Residues in an Activated State of a G Protein-coupled Receptor^*

Yong-Hun Lee, Fred Naider¶1, and Jeffrey M. Becker||2

From the Department of Microbiology and the ^||Graduate School of Genome Science & Technology, University of Tennessee, Knoxville, Tennessee 37996 and the Department of Chemistry and Macromolecular Assemblies Institute, College of Staten Island, and the ^¶Graduate School and University Center, City College of New York, New York 10314

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone -factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn²⁰⁵ as a potential interacting partner with the Tyr²⁶⁶ residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.

Received for publication, September 12, 2005 , and in revised form, November 15, 2005.

^* This work was supported in part by research grants GM22086 and GM22087 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The Leonard and Esther Kurtz Term Professor at the College of Staten Island.

² To whom correspondence should be addressed: Dept. of Microbiology, University of Tennessee, Knoxville, TN 37996. Tel.: 865-974-3006; Fax: 865-974-4007; E-mail: jbecker{at}utk.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Hauser, S. Kauffman, B.-K. Lee, F. Naider, and J. M. Becker The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding J. Biol. Chem., April 6, 2007; 282(14): 10387 - 10397. [Abstract] [Full Text] [PDF]