Abstract
Purpose:
Myocilin has two distinct cellular distributions, cytoplasmic and membrane-associated, yet appears extracellularly. We have previously shown that myocilin is released from cells on exosomes, which are a component of the endosomal pathway. In this study we tested the hypothesis that myocilin enters the exosome pathway during plasma membrane receptor endocytosis.
Methods:
Retinal pigment epithelia (RPE) in human eye-cups was stimulated with L-DOPA (1uM) to monitor endocytosis of GPR143 (OA1), an endogenous G-protein coupled receptor (GPCR) on the surface of RPE. Recombinant myocilin (WT, P370L and T377M) was heterologously co-expressed in transformed cells with GPR143 to test whether myocilin participates in receptor endocytosis and to determine the kinetics of binding. Cell surface proteins in eye-cups and transformed cells were biotinylated and protein trafficking and myocilin association were monitored over time using streptavidin beads. Finally, we tested myocilin binding to the purified cytoplasmic domain of GPR143 using chromatography.
Results:
Data obtained from both human eye-cups and cultured cells illustrate that WT myocilin binds to biotinylated plasma membrane proteins in a ligand-dependent manner. In cultured cells, peak association occurs at 20 minutes after introduction of ligand, and decreases rapidly thereafter by 60 minutes. Association of WT myocilin with GPR143, appears to occur via its cytoplasmic domain after ligand stimulation. Interestingly, the two mutant forms of myocilin exhibited different binding kinetics to biotinylated plasma membrane proteins after stimulation. P370L myocilin showed little activity, while T377M illustrated prolonged association and disturbed kinetics.
Conclusions:
Our results suggest that myocilin enters the membrane compartment of cells during receptor endocytosis. Targeting, sorting, and trafficking through this pathway is likely the mechanism by which myocilin arrives on the surface of exosomes. WT, but not mutant myocilin matched a standard time course for endocytosis. Taken together, these observations suggest that myocilin participates in receptor endocytosis, and that mutations may differentially affect receptor signaling, leading to glaucoma.
Keywords: 715 signal transduction: pharmacology/physiology •
675 receptors: pharmacology/physiology •
633 outflow: trabecular meshwork