Abstract
Purpose :
Dry eye disease affects 7 - 10% of people worldwide. Dry eye is characterized by chronic inflammation and damage of the ocular surface. Lacritin (and several of its C-terminal proteoforms) is a prosecretory growth factor in tears that, based on preclinical and cell culture studies, restores basal tearing and ocular surface health. We previously reported on identification of cornea expressed G protein-coupled receptor 87 (GPR87) as a lacritin signaling receptor out of a genome-wide CRISPR/Cas9 screen. Here we sought to validate this observation, and ask whether GPR87 couples with lacritin co-receptor syndecan-1 (SDC1).
Methods :
Validation studies of GPR87 were performed by generation of GPR87 knockout cell lines by CRISPR/Cas9 editing and by shRNA depletion of transduced human corneal epithelial (HCE-T) cells with lentiviral GPR87 sgRNA or shRNA and non-target controls. Depletion of GPR87 protein level was confirmed by western blotting. Complementation assays were performed by transducing depleted HCE-T’s with lentiviral GPR87 cDNA obtained from DNASU Plasmid Repository. 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. GPR87- lacritin binding was studied out of recombinant lacritin- or C-25-intein pulldowns. ‘C-25’, lacks lacritin's 'N-94' bioactive domain. N-94 is a synthetic peptide analogous to several lacritin C-terminal proteoforms naturally in tears.
Results :
Depletion of GPR87 abrogated N-94 rescue of IFNG/TNF stressed HCE-T’s, but was rescued by complementation with GPR87 cDNA. GPR87 complexes with SDC1 in the absence of N-94, but not with SDC1 lacking the PDZ binding domain nor lacking N-terminal domain heparan sulfate at S15 nor membrane proximal chondroitin sulfate at S184 and S185. SDC1 failed to bind GPR87 lacking the G-protein binding site “DRY” motif. Lacritin, but not C-25, binds GPR87.
Conclusions :
N-94 targets 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.
This is a 2021 ARVO Annual Meeting abstract.