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K.M. Lindsey, G.N. Magrath, A.R. Prescott, R.A. Quinlan, R.K. Crouch, K.L. Schey; Effects of Lens Aquaporin 0 Modifications on Protein–Protein Interactions . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1845.
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© ARVO (1962-2015); The Authors (2016-present)
Aquaporin 0 (APQ0), the most abundant membrane protein in the lens, functions as a water–permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The intracellular C–terminus of AQP0 is a region of interaction with other proteins. Posttranslational modifications to AQP0 occur mostly at the C–terminus suggesting potential changes in AQP0 function due to altered protein–protein interactions. Purpose: The objective of the present study is to determine how fiber cell age and modifications affect protein–protein interactions of AQP0. Methods: To examine the effect of fiber cell age on AQP0’s interaction with cytoskeletal proteins filensin and CP49, bovine lens sections were double–labeled with AQP0 antibodies and either filensin or CP49 antibodies. Fluorophore–conjugated secondary antibodies were used to visualize the labeled proteins in cortical and nuclear regions via confocal fluorescence microscopy. Immunogold–electron microscopy was also used to examine the interaction of AQP0 with filensin. To investigate the effect of AQP0 C–terminal phosphorylation on calmodulin binding, dansyl–labeled calmodulin was incubated with unphosphorylated and phosphorylated synthetic peptides mimicking AQP0 C–terminal residues 224–241. Emission spectra were acquired to examine calmodulin conformational changes upon peptide binding. A myosin light chain kinase peptide was used as a positive control and an AQP0 extracellular loop peptide was used as a negative control. Results: Immunoconfocal and immunoelectron microscopies revealed AQP0 co–localization with filensin and CP49 in cortical fiber cell membranes; however, co–localization was reduced in aged fiber cells. In addition, calmodulin bound the unphosphorylated AQP0 C–terminal peptide, while single and double phosphorylation reduced and eliminated calmodulin binding, respectively. Conclusions: The interaction of AQP0 with filensin/CP49 was reduced in the aged region of the lens where the AQP0 C–terminus is posttranslationally modified. Furthermore, phosphorylation of the AQP0 C–terminus inhibited its interaction with calmodulin. Changes in AQP0 protein–protein interactions with modification provide further insight on regulation of AQP0 function.
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