Figure 5.

Biochemical consequences of GUCY2D-LCA missense mutations in patients P1 and P2

(A) In cyto co-localization of RetGC1 mutants with GCAP1. The cDNA coding for RetGC1 tagged with mOrange fluorescent protein (red) replacing a part of RetGC1 ECD are co-expressed in HEK293 cells with bovine GCAP1 harboring enhanced GFP tag at the C-terminus (green). The rightmost panel in each row presents typical distribution of the fluorescence intensities for the two fluorochromes across the cell (scanned along the dashed lines in the cells shown in the ‘merged’ images). Upper row, wild type RetGC1; middle row, Arg588Trp; bottom row, Trp708Arg. GCAP1GFP fluorescence co-localizes with wild type RetGC1 in the ER membranes, but it fails to co-localize with Arg588Trp or Trp708Arg RetGC1 and remains uniformly distributed throughout the cytoplasm and the nucleus.

(B) Full-length wild type, Arg588Trp, and Trp708Arg RetGC1 expression in HEK293 cells was verified by Western immunoblotting. The HEK293 membrane samples containing wild type RetGC1 (n = 9 to 11 measurements), Arg588Trp (n = 3 to 4), and Trp708Arg (n = 5 to 8) were assayed for guanylyl cyclase activity (mean average ±SD) by incubating them for 30 min at 30°C in the presence (+) or in the absence (−) of 20 μM GCAP1, GCAP2, or GCAP3.