Essential role for the second extracellular loop in C5a receptor activation.
More than 90% of G protein-coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, approximately 80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor.
Klco
Jeffery M
JM
Department of Medicine, Washington University School of Medicine, Campus Box 8127, 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA.
Wiegand
Christina B
CB
Narzinski
Kirk
K
Baranski
Thomas J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D044087:Receptor, Anaphylatoxin C5a / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D003545:Cysteine