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S.I. Tomarev, M. Torrado, V.V. Senatorov, R.N. Fariss, R. Trivedi; A novel rat Pdlim2 protein interacts with actin–binding proteins in corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3806.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study possible functions of a novel Pdlim2 protein in the eye. Methods: cDNA encoding Pdlim2, a novel PDZ and Lim domain–containing protein, was identified in a cDNA library of transcripts expressed in the tissues of the rat eye angle. The expression pattern of the Pdlim2 gene was studied by northern blot and in situ hybridization. Interaction of Pdlim2 protein with other proteins was investigated in pull–down assays with corneal and lung extracts. Protein identification was performed using the nanoflow liquid chromatography and tandem mass spectrometry (LC–MS/MS) technique. Intracellular localization of Pdlim2 was investigated by confocal microscopy. Results: Rat Pdlim2 protein has a calculated molecular weight of 37.6 kDa and belongs to a family of proteins containing the PDZ domain in the N–terminal portion and LIM domain in the C–terminal portion of the protein. Pdlim2 shows 39%, 39% and 37% identity with CLP36, RIL and ALP proteins, respectively. Pdlim2 mRNA was abundantly present in the corneal epithelial cells but not in other corneal layers or in other ocular tissues. In the corneal epithelial layer, Pdlim2 mRNA was more abundant in the layer of the intermediate and superficial cells than in the less differentiated basal cells. The Pdlim2 gene was also expressed in the lung. In rat corneal extracts, α–actinin–1, α–actinin–4, filamin A, myosin VI and myosin, heavy polypeptide 9 were co–immunoprecipitated with Pdlim2. α–actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with α–actinins was confirmed using pull–down assays with purified proteins. Pdlim2 and α–actinins were co–localized with different α–actinins after transfection into COS–7 cells. In transfected COS–7 cells, complexes of Pdlim2 and α–actinin–1 were preferentially located along the basal aspect. Experiments are in progress to test direct interactions of Pdlim2 with filamin A and myosins and the role of Pdlim2 in wound healing. Conclusions: Pdlim2 interacts with several actin–binding proteins and may be involved in the modulation of the actin cytoskeleton in the cornea.
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