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Kota Gopalakrishna, Kimberly Boyd, Nikolai Artemyev; Heterologous Expression of Functional PDE6 Requires Cooperative Action of AIPL1 and the Inhibitory Pγ-subunit. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3625.
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© ARVO (1962-2015); The Authors (2016-present)
PDE6 is the effector enzyme in the phototransduction cascade and is critical for the health of photoreceptors. Heterologous expression system for PDE6 has been lacking, thus impeding the research of the mechanisms of pathogenic mutants of PDE6 and AIPL1, a presumed PDE6 chaperone. We have investigated the requirements for expression of functional PDE6 in cultured cells.
HEK293T cells were transfected with PDE6 alone and co-transfected with Pγ, AIPL1, or both. Immunofluorescence microscopy was used to study subcellular localization of the proteins. Lysates, membrane, and soluble fractions from transfected cells were prepared and examined for PDE6 protein levels and cGMP-hydrolytic activity.
cGMP hydrolysis in the PDE6 and PDE6+Pγ lysates even after removal of Pγ with trypsin did not exceed the very low levels in untransfected HEK293T lysates. PDE6 activity in the PDE6+AIPL1 lysate was markedly higher than the background level. Pγ dramatically enhanced the AIPL1-assisted expression of functional PDE6 by up to 100 fold. Mutant AIPL1 proteins linked to Leber congenital amaurosis (LCA), W72S, C89R, and C239A, completely failed to chaperone PDE6 in the heterologous system. However, several other LCA-linked AIPL1 mutants revealed partially or significantly preserved ability to fold PDE6.
AIPL1 is absolutely required for folding PDE6 into a functional enzyme. The Pγ subunit dramatically elevates expression of functional PDE6. The robust heterologous system for expression of PDE6 is developed that allows investigation of the mechanisms of mutant PDE6 and AIPL1 proteins in retinal disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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