Oral Presentation 2014 International Biophysics Congress

Identification of auxiliary sites in the relaxin/RXFP1 activation complex (#90)

Emma J. Petrie , Natalie A. Diepenhorst 1 2 , Ashish Sethi 1 , Shoni Bruell 1 2 , Samantha Lagaida 1 2 , Ross A.D. Bathgate 2 , Paul R. Gooley 1
  1. Bio21 Institute, The University of Melbourne, Melbourne, VIC, Australia
  2. Florey Neuroscience Institutes, The University of Melbourne, Melbourne, VIC, Australia

Relaxin is an emerging treatment for patients with acute heart failure, however its insulin-like two-chain structure has limited bioavailability creating a need for small molecule relaxin-mimetics

Relaxin activates a multi-domain G-protein coupled receptor (GPCR), RXFP1.  RXFP1 has a class-A GPCR transmembrane (TM) domain, but also an extracellular Leucine-rich repeat domain (LRR) and N-terminal Low Density Lipoprotein Class-A (LDLa) module tethered by linkers between the domains.

The LRRs capture circulating relaxin with high affinity.  Removal of the LDLa does not disrupt relaxin binding, however signal activaiton is abolished.  This suggests ligand binding initiates conformational changes necessary for signaling and that the LDLa is essential to this process.  We hypothesized for this to occur relaxin would make auxiliary receptor contacts in addition to the LRRs to from the active complex.

Interestingly, we can inhibit relaxin induced signaling of RXFP1 with recombinant LDLa.  For this occur we propose the LDLa makes 'ligand-like' interactions with the TM exo-loops (ELs).

We tested these hypotheses by recombinantly expressing an engineered soluble protein presenting the RXFP1 ELs -1 and -2 (ssRXFP1).  We also expressed the LDLa with the linker to the LRR domain (LDL+).  Using techniques such as circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and bio-layer interferometry (BLITZ) we characterized these proteins and showed ssRXFP1 interacts with both the LDLa and relaxin, and that relaxin also bound to the linker residues in LDL+.  Following this we probed the specificity and importance of these interactions by testing targeted RXFP1 mutants in signaling assays.

This work has allowed us to identify key residues that contribute to the RXFP1/relaxin activation complex and provides promise that these interactions can be targeted for structure-based design of RXFP1 modulators.