G protein–coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins1. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains2. Downregulation of mth increases the life span of Drosophila melanogaster3; inhibitors of Mth signaling should therefore enhance longevity. We used mRNA display selection4,5 to identify high-affinity (Kd = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane domains. Flies constitutively expressing a Mth antagonist peptide have a robust life span extension, which suggests that the peptides inhibit Mth signaling in vivo. Our work thus provides new life span–extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.
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We thank A.M. Giannetti for technical expertise on the Biacore; D.G. Myszka (University of Utah) for the SPR analysis software, Scrubber and CLAMP; M.I. Simon for use of the Flexstation automated fluorescence plate reader; T. Brummel and D. Walker for their technical expertise on the life span experiments; T.T. Takahashi and G.B. Carvalho for comments on the manuscript; and S. Cvejic and X.-Y. Huang (Cornell University Weill Medical College) for providing the HEK-Mth cell lines and details on their protocols. We are grateful to P.M. Snow (deceased, 2004) for his expertise in protein purification. This work was supported by grants from the US National Institutes of Health (R01 GM60416 to R.W.R. and R01 AG016630 to S.B.) and the Beckman Foundation (R.W.R.). W.W.J. was supported in part by a US Department of Defense National Defense Science and Engineering Graduate Fellowship, a Scholarship for Research in the Biology of Aging sponsored by the Glenn Foundation for Medical Research and the American Federation for Aging Research, and a John Douglas French Alzheimer's Foundation Postdoctoral Fellowship. A.P.W., Jr. was supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund.
The authors declare no competing financial interests.
Selected peptides recognize the full-length Methuselah receptor. (PDF 39 kb)
Amino acid analysis of selected peptides. (PDF 71 kb)
Peptide binding site is not localized to Methuselah Trp120. (PDF 56 kb)
Expression of R8-12 extends Drosophila lifespan. (PDF 127 kb)
Binding analysis of R8-04 to Methuselah by surface plasmon resonance. (PDF 62 kb)
Selected peptide sequences from Methuselah selection. (PDF 28 kb)
Crystallography data collection and refinement statistics. (PDF 34 kb)
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