Activation of the relaxin receptor RXFP1 is by a tethered agonist — ASN Events
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Activation of the relaxin receptor RXFP1 is by a tethered agonist (#47)

Paul R. Gooley 1 2 , Ashish Sethi 1 2 , Shoni Bruell 1 3 , Nitin Patil 3 4 , M. Akhter Hossain 3 4 , Daniel J. Scott 1 3 , Ross A.D. Bathgate 1 3
  1. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia
  2. Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  3. Florey Institute if Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia
  4. School of Chemistry, University of Melbourne, Parkville, Victoria, Australia

The peptide hormone H2 relaxin binds and activates the GPCR receptor, RXFP1. This receptor has a large ectodomain comprised of an N-terminal LDLa module, essential for activation (1), tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. Activation was previously thought to proceed by relaxin binding with strong affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain. In this mechanism the linker serves as a disordered region to tether the LDLa module to the receptor. However, we have found mutations within a conserved region of the linker immediately C-terminal to the LDLa module (GDNNGW, residues 41-46) to both weaken relaxin affinity and reduce activation. Using NMR spectroscopy and titrations of 15N-labelled LDLa-linker with synthetic peptide relaxin analogues we have elucidated a discrete relaxin-binding site (residues 46-63) on the linker (2).  Additional NMR experiments show residues 49-52 of the linker have a weak propensity for helix which on relaxin titration stabilizes. Mutations within GDNNGW show loss of relaxin-binding and an increase in structural disorder around this helical region of the linker, suggesting an indirect role in binding relaxin. Analysis of 15N-spin relaxation and relaxation dispersion data indicate that the helix within the linker is likely due to an interaction with the LDLa module. This interaction strengthens in the presence of relaxin, and weakens with mutations in the LDLa module previously shown to reduce activation (3). We propose two binding sites for relaxin are required: one on the LRR domain, the other on the linker. In the absence of relaxin the LDLa is in weak association with the linker, but upon binding of relaxin to the linker, the association between the LDLa module and linker is strengthened. These interactions collectively result in orienting and stabilizing structure within the GDNNGW of the linker which then functions as the tethered agonist activating the receptor.

 

  1. 1. Scott, D.J., Layfield, S., Yan, Y., Sudo, S., Hseuh, A.J., Tregear, G.W. and Bathgate, R.A.D. Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J. Biol. Chem. 2006 281, 34942-34954.
  2. 2. Sethi, A., Bruell, S., Patil, N., Hossain, M.A., Scott, D.J., Petrie, E.J., Bathgate, R.A.D. and Gooley, P.R. “The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1. Nature Communications 2016 7:11344, 1-12
  3. 3. Kong, R.C.K, Petrie, E.J., Mohanty, B., Ling, J., Lee, J.C.Y, Gooley, P.R. and Bathgate, R.A.D “The relaxin receptor (RXFP1) utilises hydrophobic moieties on a signalling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation” J. Biol. Chem. 2013 288, 28138-28151.