title: C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.
abstract: Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Baranski
authors.forename: P
authors.initials: P
authors.lastname: Herzmark
authors.forename: O
authors.initials: O
authors.lastname: Lichtarge
authors.forename: B O
authors.initials: BO
authors.lastname: Gerber
authors.forename: J
authors.initials: J
authors.lastname: Trueheart
authors.forename: E C
authors.initials: EC
authors.lastname: Meng
authors.forename: T
authors.initials: T
authors.lastname: Iiri
authors.forename: S P
authors.initials: SP
authors.lastname: Sheikh
authors.forename: H R
authors.initials: HR
authors.lastname: Bourne
mesh_terms: D000596:Amino Acids / Q000235:genetics; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics; D015936:Complement C5a / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction; D015003:Yeasts / Q000235:genetics
chemical_list: D000596:Amino Acids; D015703:Antigens, CD; D008565:Membrane Proteins; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a