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Molecular and Cellular Biology, December 1998, p. 7205-7215, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled -Factor Receptor

Peter Dube¹ and James B. Konopka^2,*

Program in Molecular and Cellular Biology¹ and Department of Molecular Genetics and Microbiology,² State University of New York, Stony Brook, New York 11794-5222

Received 22 June 1998/Returned for modification 17 July 1998/Accepted 10 September 1998

The -factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the -factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the -factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser²⁵⁴ showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln²⁵³ and Ser²⁵⁴ are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln²⁵³ and Ser²⁵⁴ fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln²⁵³ and Ser²⁵⁴ face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser²⁸⁸ and Ser²⁹²) that are potential contact residues for Gln²⁵³ because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the -factor receptor that regulates its ability to enter the activated conformation.

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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: (516) 632-8715. Fax: (516) 632-9797. E-mail: konopka{at}asterix.bio.sunysb.edu.

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Molecular and Cellular Biology, December 1998, p. 7205-7215, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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