Details

Autor(en) / Beteiligte

• Dube, Peter Herman

Titel

Genetic characterization of the activation of the G protein-coupled alpha-factor receptor

Ort / Verlag

ProQuest Dissertations Publishing

Erscheinungsjahr

1999

Link zum Volltext

• ProQuest

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The α-factor mating pheromone receptor stimulates the conjugation of the yeast S. cerevisiae. The α-factor receptor is a prototypic member of G-protein coupled receptor (GPCR) superfamily whose members are defined by possessing seven hydrophobic transmembrane domains (TMDs) and the ability to activate a heterotrimeric G protein. In order to gain insight into the molecular mechanisms of GPCR activation, a mutational analysis of the polar amino acids in TMD6 was performed. This analysis revealed that certain amino acids in TMD6 and TMD7 restrain the receptor in an inactive conformation. In particular, these results suggest a direct interaction between gln253 in TMD6 and ser288 and ser292 in TMD7. Theoretical models of GPCR structure suggests that TMD6 may also interact with TMD5. Therefore, a cysteine scanning mutagenesis of TMDs 5 and 6 was performed. This study provided genetic evidence for the interaction of conserved hydrophobic amino acids at the base of TMD5 and TMD6. Significantly, a disulfide cross-link between V223C in TMD5 and L247C in TMD6 demonstrated a physical interaction between these TMDs. Altogether, these data provide new insight into the regulation of the α-factor receptor and provide the first evidence for the orientation of TMDs 5, 6, and 7 relative to each other.