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Proteomic Identification of Preferential Interactions between PDE6 and its Inhibitory Subunit
Author(s) -
Kozacka Katie Ann,
Zhang XiuJun,
Gao XiongZhuo,
Zeng Xiaohui,
Chu Feixia,
Cote Rick H
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1052.4
Subject(s) - transducin , protein subunit , dimer , chemistry , phosphodiesterase , binding site , biochemistry , enzyme , stereochemistry , g protein , signal transduction , gene , organic chemistry
Phosphodiesterase 6 (PDE6) is central for visual transduction in rod and cone cells. PDE6 is a heterodimeric enzyme consisting of α and β catalytic subunits (Pαβ) to which two identical inhibitory subunits (Pγ) are bound (Pαβγγ). Light activation of PDE6 results from transducin binding to Pγ and relieving inhibition of catalysis. Previous studies have shown two different classes of Pγ binding sites on Pαβ, but the structural basis for the differences in binding affinity of Pγ is unknown. We hypothesized that Pγ preferentially binds to one catalytic subunit due to differences in the amino acid sequence of the two subunits. To test this, we used chemical cross‐linking of Pγ to Pαβ followed by mass spectrometric identification of which catalytic subunit was cross‐linked over a range of Pγ concentrations. Even at sub‐stoichiometric levels of Pγ relative to Pαβ, we failed to observe a statistically significant difference in Pγ binding. This lack of preferential interaction of Pγ with the catalytic subunits at sub‐stoichiometric levels suggests that binding of the first Pγ subunit allosterically reduces the affinity with which the second Pγ subunit binds to the Pαβ catalytic dimer. This may also account for biochemical evidence that transducin preferentially interacts with one of the catalytic subunits during light activation. Supported by NIH grant EY‐05798 (RHC) and NH Agricultural Experiment Station grant NH00567 (FC).