title: A role for myocilin in receptor-mediated endocytosis.
abstract: Myocilin is a broadly expressed protein that when mutated uniquely causes glaucoma. While no function has been ascribed to explain focal disease, some properties of myocilin are known. Myocilin is a cytoplasmic protein that also localizes to vesicles specifically as part of a large membrane-associated complex with properties similar to the SNARE machinery that function in vesicle fusion. Its role in vesicle dynamics has not been detailed, however myocilin intersects with the endocytic compartment at the level of the multivesicular body. Since internalized GPCRs are sorted in the multivesicular body, we investigated whether myocilin functions in ligand-dependent GPR143 endocytosis. Using recombinant systems we found that the kinetics of myocilin recruitment to biotinylated membrane proteins was similar to that of arrestin-3. We also co-localized myocilin with GPR143 and Arrestin-2 by confocal microscopy. However, wild-type myocilin differed significantly in its association kinetics and co-localization with internalized proteins from mutant myocilin (P370L or T377M). Moreover, we found that myocilin bound to the cytoplasmic tail of GPR143, an interaction mediated by its amino terminal helix-turn-helix domain. Hydrodynamic analyses show that the myocilin-GPR143 protein complex is >158 kD and stable in 500 mM KCl, but not 0.1% SDS. Collectively, data indicate that myocilin is recruited to the membrane compartment, interacting with GPCR proteins during ligand-mediated endocytosis and that GPCR signaling underlies pathology in myocilin glaucoma.
authors.affiliation: Department of Ophthalmology and Vision Science, University of Arizona, Tucson, Arrizona ; Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arrizona.
authors.forename: Brian S
authors.initials: BS
authors.lastname: McKay
authors.affiliation: Department of Ophthalmology and Vision Science, University of Arizona, Tucson, Arrizona.
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Congrove
authors.affiliation: Department of Ophthalmology and Vision Science, University of Arizona, Tucson, Arrizona.
authors.forename: Adiv A
authors.initials: AA
authors.lastname: Johnson
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina.
authors.forename: W Michael
authors.initials: WM
authors.lastname: Dismuke
authors.affiliation: Department of Ophthalmology and Vision Science, University of Arizona, Tucson, Arrizona.
authors.forename: Trent J
authors.initials: TJ
authors.lastname: Bowen
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina ; Department of Biomedical Engineering, Duke University, Durham, North Carolina.
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
mesh_terms: D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D003412:Cricetulus; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D000099179:Myocilin
chemical_list: D019393:Arrestin; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D000099179:Myocilin; C095276:GPR143 protein, human