Abstract
A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.
Highlights
Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms
Cloning and Structures of Ce-RA-guanine nucleotide exchange factor (GEF) and Hs-RA-GEF—-In a previous report [21], we carried out a yeast two-hybrid screening for C. elegans proteins associating with Ras and isolated a novel phosphoinositide-specific phospholipase C, PLC210
The deduced Ce-RA-GEF protein contained, from the N terminus to the C terminus, a cNMP-binding domain, a Ras exchanger motif (REM) domain, a PDZ domain, a RA domain and a GEF domain, all of which were predicted based on their sequence homology to the corresponding functional domains already characterized (Fig. 1, A and B)
Summary
Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms (for a review, see Ref. 1). A fragment of Hs-RA-GEF cDNA encoding amino acid residues 540 –710, encompassing the RA domain, was amplified by PCR and cloned into pMal-c (New England Biolabs, Inc.) for expression as an MBP fusion protein, MBPHs-RA-GEF-RA, in Escherichia coli.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
