A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants.
Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.
Matsumoto
Marissa L
ML
Department of Medicine and Molecular Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Narzinski
Kirk
K
Nikiforovich
Gregory V
GV
Baranski
Thomas J
TJ
D000595:Amino Acid Sequence; D015153:Blotting, Western; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
C488033:C5AR1 protein, human; D008565:Membrane Proteins; D021122:Protein Subunits; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins