Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.
The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.
Konopka
J B
JB
Department of Molecular Genetics, State University of New York, Stony Brook 11794-5222, USA.
Margarit
S M
SM
Dube
P
P
D000595:Amino Acid Sequence; D002455:Cell Division; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005799:Genes, Dominant; D000072235:Mating Factor; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009024:Morphogenesis; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010675:Pheromones / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors
D008565:Membrane Proteins; D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D011392:Proline; D019204:GTP-Binding Proteins