The human RXFP3 receptor regulates the conserved DNA damage response process

The hyper‐complex aging process is a major risk factor for progressive diseases, e.g . Alzheimer's disease, atherosclerosis and Type II diabetes. ‘Pathological’ aging and these age‐related disorders demonstrate a considerable functional overlap, i.e . progressive decline of cell stress resilience, energy metabolism dysfunction and oxidative DNA damage. Complex pathologies such as aging likely involve the coordination of multiple signaling pathways. The protein factors that facilitate communication between the subnetworks are termed ‘keystone’ proteins. These proteins coordinate responses to dynamic molecular events through protein‐protein interaction organization, binding to multiple partners, allowing bridging/connecting of multisystem processes. We have previously identified one such ‘aging keystone’, GIT2 (G protein‐coupled receptor kinase interacting protein 2). As a scaffold protein GIT2 is not a classical drug target. To identify a potential therapeutically‐targetable factor, e.g . G protein‐coupled receptor (GPCR), that controls GIT2 expression/function we investigated co‐expression variance in tissues from GIT2 knockout (GIT2KO) mice. Unbiased transcriptomic analysis of these tissues, demonstrated a consistent co‐expression relationship of GIT2 with Relaxin family peptide receptor 3 (RXFP3). Interestingly these two proteins demonstrate considerable functional overlaps related to metabolic aging patterns, e.g . both proteins play important roles in cell stress resilience and energy metabolism. To further connect these two factors, within an aging context, we investigated whether, like GIT2, RXFP3 was associated with oxidative stress or DNA damage reponse (DDR). Hence we demonstrated that like GIT2, RXFP3 physically responded to oxidative stress, showing a translocation from the plasma membrane towards the nucleus and cytoskeleton. This reaction, in absence of the cognate peptide ligand (relaxin 3 – RLN3), indicates that this receptor, like GIT2, may be an oxidative stress sensor involved in DDR processes. Expanding this finding we analyzed the effect of oxidizing and DNA‐damaging stressors on the binding partners of RXFP3 using SILAC‐based affinity‐purification mass spectrometry (AP‐MS). Investigating the stress‐induced binding partners of RXFP3 showed the presence of multiple proteins linked to DDR and cell cycle control. Extending our investigation we next found that i) RXFP3 expression is increased in response to DNA damage agents and ii) overexpression, or RLN3‐mediated stimulation, of RXFP3 attenuates DNA damage, evidenced by reduced yH2AX, ATM and BRCA1 phosphorylation. The functional synergies shown by RXFP3 and GIT2, with respect to stress sensation and DDR functions encouraged us to believe that this receptor‐signaling protein system may form a crucial signaling system that regulates cellular degradation after DNA damage and/or aging, and thus represents a novel strategy to attenuate the rate of age‐related damage accumulation. Support or Funding Information This work was supported by the FWO‐OP/Odysseus program (42/FA010100/32/6484), FWO Travelling Fellowship Program (V4.161.17N), GOA progam, BOF (33931) and Erasmus+.Visualization of the stress sensor Relaxin Family Peptide 3 receptor (RXFP3) interactome analysis after affinity‐purification mass spectrometry (AP‐MS). We analyzed the interacting proteins for RXFP3 after causing oxidative DNA damage in cells. Only proteins showing a selective binding (expression ratio >1.5) were analyzed using natural language processing giving a clear appreciation of our data set. Here we see that RXFP3 interacts with proteins involved in DNA damage response processes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .