Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-06, Vol.93 (13), p.6764-6769
• Main Authors: Konopka, J.B. (State University of New York, Stony Brook, NY.), Margarit, S.M, Dube, P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biochemistry; Cell Division; CHIMIORECEPTEUR; ESTIMULO; FEROMONAS; Gene induction; Genes, Dominant; Genetic mutation; GTP-Binding Proteins - metabolism; Ligands; Mating Factor; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Molecular Sequence Data; Morphogenesis; MUTACION; Mutagenesis; MUTANT; MUTANTES; MUTATION; NUCLEOTIDE; NUCLEOTIDOS; Peptides - metabolism; PHEROMONE; Pheromones - metabolism; Plasmids; PROLINA; PROLINE; Proline - genetics; Proline - metabolism; Protein Binding; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; Proteins; QUIMIORECEPTORES; Receptors; Receptors, Mating Factor; Receptors, Peptide - genetics; Receptors, Peptide - metabolism; Reporter genes; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Signal Transduction; STIMULUS; Transcription Factors; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.13.6764

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 leads to 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 greater than or equal to 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