Novel GPR87/SDC‐1 Complex Modulates Lacritin Rescue of Homeostasis

Abstract

IntroductionOcular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world’s population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a prosecretory mitogenic tear and plasma glycoprotein, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFNG and TNF stress. It does so by transiently accelerating epithelial autophagy (FOXO1/ATG7; FOXO3/ATG101) thereby restoring oxidative phosphorylation, as validated in a recent 204 patient phase 2 clinical trial of human Sjögren's Syndrome Dry Eye. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Here we report the identification of G protein‐coupled receptor 87 (GPR87) as the lacritin signaling receptor out of a genome‐wide CRISPR/Cas9 death screen using the C‐terminal lacritin synthetic peptide N‐94 as probe. GPR87 is expressed in the cornea and has a critical role in cellular proliferation and survival. Years ago, GPR87 was thought to be an LPA receptor, later disproven by Arfelt et al, ′17. Finally, we also report that GPR87 interacts with syndecan‐1 (SDC1), a cell surface co‐receptor for lacritin. We observed a direct binding of recombinant lacritin to GPR87, corroborating with the hypothesis that GPR87 is a critical receptor for lacritin homeostatic signaling.MethodsValidation studies of GPR87 were performed by generation of GPR87 knockdown cell lines by shRNA depletion by transducing HCE‐T cells with lentiviral GPR87 shRNA and non‐target controls. Depletion of GPR87 protein level was confirmed by western blot. Complementation assays were performed by transducing depleted HCE‐T’s with lentiviral GPR87 mouse cDNA. Lacritin affinity binding to GPR87 was first studied out of recombinant lacritin‐ or lacritin C‐25‐intein pulldowns. Truncation mutant ‘C‐25’, lacks lacritin's ‘N‐94’ bioactive domain. Lacritin pulldowns were challenged using each of eleven synthetic peptides corresponding to or together spanning all eight extracellular or intracellular strands or loops of GPR87 ‐ each at increasing concentrations (20, 100, 400 µM). To explore whether GPR87 can couple with SDC1, we blotted for GPR87 out of SDC‐1 pulldowns from HEK2936E cells co‐transfected with wild type or mutated GPR87 and SDC1 cDNA’s.ResultsLacritin, but not C‐25, binds GPR87 via GPR87's outer three loops with outer loop 3 peptide most inhibitory. GPR87 complexes with SDC1 in the absence of N‐94, but not with SDC1 lacking the PDZ binding domain nor with GPR87 lacking the G‐protein binding site “DRY” motif. SDC‐1 lacking N‐terminal domain heparan sulfate at S15 or membrane proximal chondroitin sulfate at S184 and S185 failed to bind GPR87. SDC‐1 also failed to complex with GPR87 in cells treated with chlorate to suppress sulfation.ConclusionsLacritin appears to bind directly to the outer loop domains of GPR87 as part of a preformed SDC1‐GPR87 complex ‐ the latter linked in part by glycosaminoglycans and apparently by cytoplasmic SDC1 and GPR87 domains to regulate ocular surface health.