Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotidegated channel by the tyrosine kinase adapter protein Grb14

Abstract

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In this study we show that this RA domain mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg559 through a negatively charged β- turn at its RA domain. Our study shows that the three Glu residues (180–182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our studies also suggest that substitution of Lys140 with Ala or in combination with polyglutamte mutants of Grb14 resulted in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu180–182 and Lys140 other residues in Grb14 may likely to be involved in the electrostatic interaction with CNGA1. The RA-domain is highly conserved among the members of Grb7 family of proteins which includes Grb7, Grb10 and Grb14. Our data indicate that Grb14 only is able to modulate the channel activity, but not Grb7 and Grb10. These studies suggest the existence of a divergence in RA domains among the members of the Grb7 family.

Conservation and divergence of Grb7 family of Ras-binding domains

“…Consistent with these results we recently reported that Grb14 only is able to modulate the channel activity, but not Grb7 and Grb10 (Gupta et al, 2011). These results suggest the existence of a divergence among the members of the Grb7 family, and that Grb14 might have been evolved later in the evolution that binds to Ras and nucleotide binding proteins.…”

“…Further studies are required to examine the release of bound Grb14 from ROS at different concentration of cGMP and also to study the cGMP-mediated translocation of Grb14. Very recently we reported that Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg 559 through the negatively charged Glu residues (180–182) on β-turn at its RA domain which results in the inhibition of channel activity (Gupta et al, 2011). Our studies suggest that Grb14 is a novel physiological modulator of CNG channel and we found that channel is more sensitive to cGMP or opens at a lower concentration of cGMP in Grb14 −/− mice (Gupta et al, 2010).…”

“…Myc-tagged bovine Grb14, Grb10 and Grb7 constructs have been described previously (Gupta et al, 2011). The RA (residues 105–195) domains of Grb14 was cloned into Myc-tagged pCDNA3 vector as described previously (Gupta et al, 2010).…”

“…Recently a novel function of an RA domain containing protein in photoreceptors has emerged from our studies (Gupta et al, 2010). We found that RA-domain of Grb14 interacts with the nucleotide binding pocket of a cyclic nucleotide gated channel alpha subunit (CNGA1) and inhibits its activity (Gupta et al, 2010; Gupta et al, 2011). It is interesting to note that CNGA1 is not a Ras protein; however, both Ras and CNGA1 are nucleotide binding proteins.…”

Insulin receptor regulates photoreceptor CNG channel activity

“…We have reported previously that Grb14, an insulin receptor (IR)-binding protein, directly binds to the COOHterminal region (CTR) of CNGA1 subunit and inhibits channel activity in a light-dependent manner (11). The interaction is mediated through a Ras-associating domain of Grb14 binding to Ras-like domain in CNGA1 (11,12,36). Our studies also suggest that Grb14 is a competitive inhibitor of cGMP, and thereby it facilitates the closure of the channel to an off state (11).…”