C5a receptor activation - Genetic identification of critical residues in four transmembrane helices

Hormones and sensory stimuli activate serpentine receptors, transmembrane s witches that relay signals to heterotrimeric guanine nucleotide-binding pro teins (G proteins). To understand the switch mechanism, we subjected 93 ami no acids in transmembrane helices III, V, VI, and VII of the human chemoatt ractant C5a receptor to random saturation mutagenesis. A yeast selection id entified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix sur faces 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 s ubstitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct compon ents of the receptor switch, a surface at or near the extracellular membran e interface and a core cluster in the cytoplasmic half of the bundle. Twent y-one of the 121 mutant receptors exhibit constitutive activity, Amino acid s substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellula r end of helix VI. These results identify key elements of a general mechani sm for the serpentine receptor switch.