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. 2011 Jul 15;22(14):2509–2519. doi: 10.1091/mbc.E11-02-0157

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© 2011 Liu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — KRIT1 is a Rap1-specific effector protein, and its interaction with Rap1 requires the F1 subregion of the FERM domain and the switch I domain of Rap1. (A) Recombinant bead-bound GST-Rap1A, GST-H-Ras, and GST were used to isolate recombinant KRIT1 and RalGDS from cell lysates. KRIT1 binds to Rap1A but not H-Ras or GST, whereas RalGDS binds to both Rap and Ras proteins (left). KRIT1 preferentially binds active Rap1A (GST-Rap1V12) and does not bind to an inactive Rap1 variant (GST-Rap1N17) or to GST only (right). GST blot is shown as loading control. Blots are representative; n = 3. Blots were cropped, and intervening lanes were removed using Adobe Photoshop. (B) KRIT1–Rap1A interaction involves residues within the switch 1 domain of Rap1A. Recombinant Rap1A (V12) from HEK293 cell lysate binds to GST-F123 (left). Single-alanine mutation on a switch 1 domain residue D38 (Rap1V12A38) does not bind to GST-F123 (right). Another switch 1 domain mutant (Rap1V12A37) dramatically reduces the binding to GST-F123 (middle). Blots are representative of three experiments. (C) KRIT1 binding to Rap1A is GTP dependent. Recombinant Rap1A from cell lysate binds to the KRIT1 FERM domain (GST-F123) only in the presence of GTP (10 mM). Binding of Rap1A to GST-KRIT1 FERM is lost on deletion of the F1 region, as binding to truncated FERM domain protein (GST-F23) is not observed (top). Expression of GST constructs is shown at bottom; blots are representative of three experiments. (D) F1 subregion of KRIT1 FERM domain is sufficient for Rap1A binding. Top, recombinant Rap1A (V12) binds to GST-F1 (right) but not GST alone (left). Bottom, equal loading of both GST fusion proteins as judged by SDS–PAGE and Coomassie Blue staining. Blots are representative of three experiments.