Figure 2.

IQSEC2 structure and mutations. (a) Scheme of the human IQSEC2 protein (NP_001104595); shown are the IQ-like domain, Sec7 domain, pleckstrin homology domain (PH) and PDZ binding motif (UniprotKB/Swiss-Prot for Q5J85;1,488-amino-acid protein). Mutations in each family are shown above. (b) Missense mutations introduced into the Sec7 domain (light gray) diminish the GTP binding activity compared to the Sec7 wild-type (black) activity to levels seen with the E849A dominant negative mutant (dark gray) predicted to reduce GEF activity of the Sec7 domain. An all-groups comparison of the mean pMol of GTP bound to ARF6 was achieved by linear mixed model analysis. *P < 0.0001 compared to Sec7Wt. (c) Wild-type (Wt) ARF6-HA and Flag-tagged wild-type or mutant IQSEC2 were transfected into HEK293 cells and lysates were subjected to a pull-down assay with GST:GGA3 to isolate ARF6-GTP. The precipitates (above, top row) and lysates (above, bottom row) were probed with anti-HA to detect ARF6 and anti-Flag to detect expression of IQSEC2 (above, middle row). ARF6-GTP levels were normalized to total ARF6 levels and shown as the fold-increase over ARF6 transfected with empty vector. The E849K dominant negative mutation abolishes GEF activity of the Sec 7 domain⁸. Data are mean ± s.e.m. from three independent experiments (bottom panel). (d) The IQ-like motif sequence lacks the G and second basic residue of the IQ motif. Characters within parentheses can substitute for each other. The R359C mutation disrupts the basic (R) residue (in bold and boxed).