The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor.
The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.
Parrish
William
W
Department of Molecular Genetics and Microbiology, Center for Structural Biology, State University of New York, Stony Brook, New York 11794-5222, USA.
Eilers
Markus
M
Ying
Weiwen
W
Konopka
James B
JB
D019204:GTP-Binding Proteins / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D019204:GTP-Binding Proteins