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Updated: May 11, 2026

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Selectivity of CDC25 homology domain-containing guanine nucleotide exchange factors
Milica Popovic1, Marije Rensen-de Leeuw, Holger Rehmann
1Molecular Cancer Research, Centre of Biomedical Genetics and Cancer Genomics Centre, University Medical Center Utrecht, Utrecht, The Netherlands.
Abstract:
The Ras family of small G-proteins plays an essential role in the regulation of a variety of signal transduction processes, ranging from cell cycle control to the regulation of exocytosis. Signalling by the Ras G-proteins is initiated by the CDC25 homology domain (CDC25-HD) containing guanine nucleotide exchange factors (GEFs); each GEF, with its specific selectivity profile towards G-proteins, commonly acts on only a small subset of the Ras family members. Thus, GEFs play a pivotal part in establishing the activation of the downstream signalling routes. The structural basis for the establishment of selectivity in the GEF-G-protein interaction is only partially understood, and several controversies on the selectivity of GEFs are discussed in the literature. In the present study, we undertook a systematic approach to determine the selectivity of CDC25-HD for members of the Ras family. We generated a data set of 126 pairs using a standardised in vitro approach encompassing purified recombinant proteins, and a comprehensive mutational study analysed the basis of the selectivity. Together, these data highlight the distinct selectivity of various GEFs and allow for predictions of untested combinations of GEFs and G-proteins.
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