Figure 2

Parafibromin and actinin interacting regions. (A) Schematic of the 531 amino acid (aa) parafibromin protein. Striped regions are the 3 nuclear localization signals (aa 76–92, 125–139, and 393–409). The 2 black lines on top show the β-catenin interacting region (aa 218–263), and the Cdc73p homologous region (aa 343–531). Below are shown regions of parafibromin fused to the Gal4DBD that were tested for interaction with Gal4AD-actinin-3 by yeast two-hybrid assays as described in the footnote of Table 2. (B) Schematic of actinin-3 protein (901 amino acids). Following the N-terminal actin-binding region are 4 striped areas corresponding to the 4 spectrin repeats: SR-1 (aa 288–398), SR-2 (aa 408–512), SR-3 (aa 523–634), and SR-4 (aa 642–747). The two black areas near the C-terminus are the EF-hand domains (aa 764–793, and 800–828). Below are shown regions of actinin-3 fused to the Gal4AD that were tested for interaction with Gal4DBD-parafibromin by yeast two-hybrid assays as described in the footnote of Table 2. The dashed grey shaded area (aa 330–397) in the first actinin-3 construct corresponds to the location of the truncated N-terminus (aa 323, 327, 329, 333, 340, 360, 370, 377, 381 and 390) of the 10 independent actinin-2 clones, in-frame with the Gal4AD, isolated by yeast two-hybrid library screening. Also shown is the schematic of actinin-1 protein (892 amino acids) representing the two non-muscle actinins (actinin-1 and actinin-4) that do not interact with parafibromin. They show over 75% amino acid sequence identity with the muscle actinins (actinin-2 and actinin-3). Their domain composition and domain location is similar to the muscle actinins. However, the muscle actinins possess amino acid substitutions (not shown) in the EF hand domains that prevent calcium binding and render them insensitive to calcium. Calcium binding reduces the affinity of non-muscle actinins for F-actin [35]. Interaction is indicated by β-galactosidase activity level. -, +, ++, or +++ indicate no, low, medium or high levels of β-galactosidase activity, respectively. Activity level intermediate between 2 categories is indicated by a slash separating the 2 categories.