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J Biol Chem

Title:		Interaction between transmembrane domains five and six of the alpha -factor receptor
Author(s):		Dube P; DeCostanzo A; Konopka JB;
Address:		"Program in Molecular and Cellular Biology and the Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222, USA"
Journal Title:		J Biol Chem
Year:		2000
Volume:		275
Issue:		34
Page Number:		26492 - 26499
DOI:		10.1074/jbc.M002767200
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae. Previous studies implicated the third intracellular loop of this receptor in G protein activation. Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified. Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes. Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6. Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact. In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor. Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling"
Keywords:		"Amino Acid Sequence Amino Acid Substitution Cysteine Membranes Molecular Sequence Data Mutagenesis, Site-Directed Phenotype Protein Structure, Secondary Receptors, Mating Factor Receptors, Peptide/*chemistry/genetics Structure-Activity Relationship Transc;"
Notes:		"MedlineDube, P DeCostanzo, A Konopka, J B eng R01 GM055107/GM/NIGMS NIH HHS/ GM55107/GM/NIGMS NIH HHS/ T32CAO9176/CA/NCI NIH HHS/ Research Support, U.S. Gov't, P.H.S. 2000/06/10 J Biol Chem. 2000 Aug 25; 275(34):26492-9. doi: 10.1074/jbc.M002767200"