Genetic mapping of the human C5a receptor. Identification of transmembrane amino acids critical for receptor function.
Many hormones and sensory stimuli signal through a superfamily of seven transmembrane-spanning receptors to activate heterotrimeric G proteins. How the seven transmembrane segments of the receptors (a molecular architecture of bundled alpha-helices conserved from yeast to man) work as "on/off" switches remains unknown. Previously, we used random saturation mutagenesis coupled with a genetic selection in yeast to determine the relative importance of amino acids in four of the seven transmembrane segments of the human C5a receptor (Baranski, T. J., Herzmark, P., Lichtarge, O., Gerber, B. O., Trueheart, J., Meng, E. C., Iiri, T., Sheikh, S. P., and Bourne, H. R. (1999) J. Biol. Chem. 274, 15757-15765). In this study, we evaluate helices I, II, and IV, thereby furnishing a complete mutational map of the seven transmembrane helices of the human C5a receptor. Our analysis identified 19 amino acid positions resistant to non-conservative substitutions. When combined with the 25 essential residues previously identified in helices III and V-VII, they delineate two distinct components of the receptor switch: a ligand-binding surface at or near the extracellular surface of the helix bundle and a core cluster in the cytoplasmic half of the bundle. In addition, we found critical amino acids in the first and second helices that are predicted to face the lipid membrane. These residues form an extended surface that might mediate interactions with lipids and other membrane proteins or function as an oligomerization domain with other receptors.
Geva
A
A
Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Lassere
T B
TB
Lichtarge
O
O
Pollitt
S K
SK
Baranski
T J
TJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D002874:Chromosome Mapping; D005656:Fungal Proteins; D015723:Gene Library; D005838:Genotype; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D015703:Antigens, CD; D005656:Fungal Proteins; D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement