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G. Hoppe, A. Sears, J. E. Sears; Molecular Basis for Rapid Corticosteroid Effects: Isolation of Plasma Membrane Glucocorticoid Receptor from Retinal Glia. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3725.
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To identify the alternative pathway of for anti-inflammatory and anti-angiogenic effects of glucocorticoids (GC). GC reduce neovascularization and resolve retinal edema through one of the two distinct mechanisms, the "classic" GC nuclear receptor or an unconventional membrane-associated receptor that initiates transcription-independent cell response. We hypothesize that this non-genomic GC receptor in retinal glia is responsible for rapid destabilization of key mRNA of the inflammatory and angiogenic factors.
Isolation of GC-binding proteins from human Müller cells (MIO-M1) was achieved by using cortisol tagged with biotin and a photocrosslinker. Cellular membrane fractions were obtained by sequential extraction into non-denaturing detergent cocktails. Following magnetic streptavidin affinity purification and separation by SDS-PAGE, GC-bound proteins were detected by anti-biotin antibody and identified by mass spectrometry and bioinformatics.
Optimization of crosslinking/extraction/purification protocol yielded consistent protein band patterns on 1D PAGE, yet biotinylated cortisol was competed out by an excess of unlabeled ligand from only two bands, 65 kDa and 250 kDa. To further reduce the complexity of protein mixtures we performed 2D PAGE, which was able to resolve only the 65 kDa spot that was consequently identified as cytoskeleton-associated protein 4 (CKAP4). CKAP4 is a single-pass protein found in endoplasmic reticulum and plasma membranes and is ubiquitously expressed in many tissues. CKAP4 is a receptor for tissue plasminogen activator, a receptor for antiproliferative factor and a receptor for surfactant protein. The sole previously-identified binding partner of CKAP4, ASCC2 is implicated multiple nuclear receptor pathways including NF-kB, estrogen receptor, and AP1.
A GC-specific biotin-photocrosslinker helped to identify novel cortisol-binding membrane protein implicated in inflammatory and stress pathways. This discovery opens the possibility of developing therapies for inflammation, vascular permeability and neovascularization that provide the benefit of GC action without the debilitating side effects of prolonged steroid use.
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