Non-canonical cleavage of GPR125/ADGRA3 releases extracellular receptor fragments detectable in cerebrospinal fluid.

Adhesion G protein-coupled receptors (aGPCRs) are crucial for barrier tissues and typically autoproteolytically cleaved into N- and C-terminal fragments (NTF and CTF) that remain non-covalently associated until separation is triggered. Here, we show that human GPR125 (ADGRA3), expressed in choroid plexus epithelium, is not cleaved at the GPCR autoproteolysis site (GPS), consistent with its divergent SLS motif. Instead, it undergoes non-canonical proteolytic processing that generates and releases atypical, non-canonical N-terminal fragments (ncNTFs), a process further enhanced by insulin. Using mutational analyses, metalloprotease inhibition, and ADAM10/17-deficient cells, we find that multiple proteases likely participate in cleavage. Mass spectrometry reveals one specific cleavage site, and real-time imaging uncovers GPR125's intracellular trafficking and shedding dynamics. Changing the GPS destabilizes the ncNTF-ncCTF association and increases ncNTF release, thereby promoting cell viability. Finally, detection of multiple GPR125-derived peptides in human cerebrospinal fluid suggests ncNTF shedding in vivo and possible biological relevance.