Investigating Potential Extracellular Receptors for REG3A on Peripheral Axons

Neurons extensively use post-transcriptional regulation to fine tune gene expression and this process is driven by RNA binding proteins (RBPs). The RBP KHSRP regulates the stability of messenger RNAs (mRNAs) by binding directly to AU-rich elements (ARE) in 3’ untranslated regions (UTR) of mRNAs. Depletion of KHSRP from neurons increases axon and dendrite growth during development, indicating that KHSRP has a growth inhibitory role. In searching of signals that might regulate Khsrp mRNA translation in regenerating axons, earlier work from the Twiss lab showed that Reg3a mRNA localizes in peripheral nerve axons 3 days after nerve injury. We have since found a corresponding increase in axonal REG3A protein and a concordant increase in Khsrp mRNA translation with recombinant REG3A (recREG3A) treatment. Based on these observations, we hypothesize autocrine signaling of secreted REG3A activates an increase in intra-axonal calcium. That increased calcium activates axonal translation of KHSRP and slows nerve regeneration. REG3A likely binds to a cell surface receptor to mediates downstream signaling and elevation of calcium, but the identity of such protein(s) in neurons is not clear. To identify the potential neuronal REG3A receptor, we performed binding and extracellular cross linking with HIS-tagged recREG3A, followed by affinity isolation and mass spectrometry. Curating the mass spectrometry hits for membrane proteins showed CKAP4, TENM3, SOGA3, ITGB4, RTN4, and NRP1 among the top hits. These potential REG3A receptors were systematically assessed by siRNA depletions and analyzing responsiveness of dorsal root ganglion (DRG) cultures to recombinant REG3A. These initial studies have developed a platform for functionally assessing REG3A signaling based changes in axonal Ca2+, Khsrp mRNA translation, and axon growth.