Regulation Of GTP-binding Proteins

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ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere). ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs), some of which are inhibited by BFA (a drug that inhibits protein secretion and causes reversible disintegration of Golgi cisternae) and some of which are BFA-resistant. Among the latter is the family of ~50-kDa cytohesins. In addition to the ~200-amino acid Sec7 domain present in all GEPs, the cytohesin molecule contains a PH domain that is responsible for phospholipid binding, and a small coiled-coil domain at the N-terminus. Three cytohesins were known when identification and characterization of cytohesin-4 by the group led them to compare gene structures of cytohesin-1 and cytohesin-4. This resulted in their demonstration that alternative splicing of transcripts of cytohesin-1, -2, and -3 produces mRNAs differing by a single codon. The resulting protein isoforms contain either two or three consecutive glycines near the N-terminus of the PH domain, with important functional consequences for the specificity of interaction with phosphatidylinositol phosphates. In recent collaborative studies, microarray technology was used to evaluate effects of IL-2 and IL-12 on gene expression in Jurkat T cells. Increased expression of a 1.3-kb mRNA identified in the data base, as a cytohesin-binding protein was confirmed by incubation of peripheral blood mononuclear cells with IL-2 and/or IL-12. The protein product termed Cybr (cytohesin binder and regulator) contains 359 amino acids, including a PDZ domain and a coiled-coil domain at the C-terminus. The interaction of Cybr and cytohesin-1 required the coiled-coil domains of both proteins. In preliminary experiments, enhancement of cytohesin activity by Cybr was observed only over a very narrow range of protein concentrations, perhaps related to the potential homodimerization of cytohesin, which also involves the coiled-coil domain. Two BFA-inhibited GEPs (BIG1 and BIG2) had been purified by the group as components of a ~670-kDa macromolecular complex. Subsequent cloning was followed by structure-function studies with recombinant proteins. Immunoprecipitates of endogeneous BIG1 and BIG2 with specific antibodies precipitated also 70-75% of the other protein. Analyses of other components of immunoprecipitaes and of the conditions that modify their composition are continuing to define how these two BFA-inhibited GEPs are regulated, independently or coordinately.