Localization and functional importance of GPR125 in the liver

Abstract

Of all of the protein families in the human genome, G protein-coupled receptors (GPCRs) comprise the largest, representing approximately 3-4% of all human genes. To identify high-expressing GPRs with unknown functions in the liver, we performed a TaqMan array screen using both male and female C57BL6 livers with expression normalized to GAPDH. Amongst others, we identified high expression of adhesion GPCR (aGPCR) GPR125 whose ΔCt value was 17.2. As a comparison, the Glucagon receptor - which plays an essential role in liver glucose homeostasis - had a similar ΔCt value of 16.0 supporting the importance of furthering our understanding of GPR125. aGPCRs are classified by their unique N-terminal domain containing, among others, a GPCR auto proteolysis-inducing (GAIN) domain which contains the GPS cleavage site. The presence of this domain, ubiquitous among almost all aGPCRs, facilitates the cleavage and non-covalent attachment of the N-Terminal (NTF) and C-Terminal Fragments (CTF). When the NTF is displaced (via mechanosensation or protein-protein binding), this is thought to expose a tethered ligand leading to GPCR activation. However GPR125 is atypical, as the GPS site does not contain the classical His-Leu/Thr sequence (where cleavage occurs between the leucine and threonine) that is thought to be necessary for cleavage, and instead contains an Ser-Leu/Ser sequence. Despite this, recent studies have shown that cleavage can occur, although whether it contains a tethered agonist has yet to be investigated. To test this, synthetic peptides corresponding to the β-strand immediately following the predicted GPS domain were synthesized and tested for activation using the PRESTO-TANGO assay, a luminescence readout to indicate receptor activation. Indeed, we determined that GPR125 is activated by a tethered ligand with maximal activation detected with the complete β-strand and 2 additional c-terminal amino acids. Notably, when the peptide was lengthened, we observed a loss of activation suggesting that the maintenance of the helical structure is critical for GPR125 activation. By using RNA scope, an in-situ hybridization assay, we localized GPR125 to the cuboidal endothelial cells in the lining of the bile duct, as well as some generalized localization to hepatocytes. This localization was confirmed through antibody staining which revealed that GPR125 localizes to the basolateral membrane of cholangiocytes. This localization, coupled with previous findings in the Zebrafish model, suggest that GPR125 may play a critical role in liver development and repair following bile duct injury. Efforts are currently underway to test this using an adenoviral delivery of CRISPR-Cas9-GPR125 guide RNAs to generate a liver-specific knockout. NIDDK and NIH Common Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.