Figure 7
Molecular determinants involved in RA-PI3Kγ interaction with CNGA1. Ribbon-shaped model of the RA domain of Grb14 [UniProtKB Accession No. Q5ICW4] (106–192) constructed using the RA domain of Grb10 as a template (PDB ID: 3HK0). The interacting residues of RA-Grb14 (A) and RA-PI3Kγ (B) are highlighted as ball and stick (red) and labeled. The corresponding secondary structural motifs of the two structures are similarly colored. Complete view of the interaction between RA-PI3Kγ (ribbon, multicolored) and the cytoplasmic domain of CNGA1 (CPK, blue; C). The positively charged random coil element of RA-PI3Kγ with projecting Lys side chains (251 pink, 255 brown, 256 purple and 254 red; ball and stick, labelled) interacts with Asp577 (green space-filled and labeled), Asp578 (gray space-filled and labeled) and Glu581 (light blue, space-filled and labelled). Glu488 (yellow, space-filled and labeled) provides additional anchorage through the interaction with K254. The interacting residues of RA-PI3Kγ are highlighted as ball and stick and labeled, while the CNGA1 interacting residues are represented as space-filled structures and labeled. We investigated the interaction between RLD-CNGA1 and its mutant proteins by subjecting the HEK-293 T cell lysates expressing respective proteins to immunoprecipitation with anti-Flag antibody (D), followed by immunoblotting and probing for RA-PI3Kγ with anti-Myc antibody (D, F) and vice versa (E, G). Cell lysates were also loaded in each case for comparison. The interaction was quantified by densitometric analysis of the band intensities in immunoblots, performed in the linear range of detection, and normalized by the total amount of RLD-CNGA1 or RA-PI3Kγ immunoprecipitated in each case (H). The relative values were expressed as percentage of the co-immunoprecipitation in wild-type proteins. Data are mean ± SD, n = 3, significance between each of the wild type RLD-CNGA1, D577N, E581Q, D577-578 N, and DDE 577,578, 581 NNQ *p < 0.001.