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V. K. Gupta, A. Rajala, R. V. Rajala; Unexpected Binding and Modulation of Photoreceptor Specific Cyclic Nucleotide Gated Channel Function by Grb14 Through Its Ras-Associating Domain. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1084.
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Growth factor receptor-bound protein 14 (Grb14) is an adapter protein involved in the insulin receptor signaling. We recently reported that the intracellular localization of Grb14 is light-dependent. The translocation of Grb14 requires rhodopsin photoactivation, but not signaling through the phototransduction cascade, and is not based on direct Grb14-rhodopsin interactions. We identified photoreceptor cGMP-gated channel A1 (CNGA1) as an interaction partner of Grb14 in the outer segments. The mechanism of interaction with Grb14 and functional aspects of the channel modulation are examined here.
Glutahione S-transferase pull-down, membrane yeast two-hybrid assays, immunoprecipitation and immunoblot techniques were employed to study the in vivo and in vitro interaction between CNGA1 and Grb14. The functional role of Grb14 on CNGA1 channel modulation was measured through cGMP induced Ca2+ permeability of expressed CNGA1 channel in heterologous system. Molecular modeling followed by site-directed mutagensis was employed to study the mechanism of interaction and modulation of channel activity by Grb14.
In light-adapted conditions, we identified the in vivo interaction between CNGA1 and Grb14. We found the inhibition of CNGA1 channel activity by Grb14. The channel modulation is dictated by direct binding of the Grb14 Ras-associating domain with CNGA1. The interaction is mediated through Glu residues on the surface of Grb14 (E180-182) which are able to interact with Arg559 in the cGMP binding pocket of CNGA1 and modulate its activity.
The functional consequence of this interaction is that in light, Grb14 binds to CNGA1 and facilitates the closure of CNG channel. This study demonstrates for the first time that Grb14 regulates light adaptation and provides molecular evidence that Grb14 is a modulator of CNG channel function.
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