Strategies for isolating constitutively active and dominant-negative pheromone receptor mutants in yeast.
Mating pheromone receptors of the yeast Saccharomyces cerevisiae are useful models for the study of G protein-coupled receptors. The mating pheromone receptors, Ste2 and Ste3, are not essential for viability so they can be readily targeted for analysis by a variety of genetic approaches. This chapter will describe methods for identification of two kinds of mutants that have been very informative about the mechanisms of receptor signaling: constitutively active mutants and dominant-negative mutants. Interestingly, these distinct types of mutants have revealed complementary information. Constitutive signaling is caused by mutations that are thought to weaken interactions between the seven transmembrane domains (TMDs), whereas the dominant-negative mutants apparently stabilize contacts between TMDs and lock receptors in the off conformation. In support of these conclusions, certain combinations of constitutively active and dominant-negative mutants restore nearly normal signaling properties.
Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, CSIC-University of Salamanca, Campus Unamuno, Salamanca, Spain.
Mercedes
M
Dosil
James B
JB
Konopka
D005822:Genetic Vectors / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D051964:Receptors, Mating Factor / Q000235:genetics* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification*; D015398:Signal Transduction; D014170:Transformation, Genetic
D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; C081863:STE3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins