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 sectretion and causes reversible disintegration of Golgi cisternae) and some of which are BFA-resistant. Among the latter is the family of ~50-kDa cytohesins, the fourth member, of which was cloned and characterized by the group. Two BFA-inhibited GEPs (BIG1 and BIG2) had been purified by the group as components of ~670-kDa macromolecular complex. Subsequent cloning was followed by structure-function studies with recombinant proteins. Immunoprecipitates of endogeneous BIG1 and BIG2 from cultured cells with specific antibodies precipitated also 70-75% of the other protein. The two very similar BIGs behaved quite differently in yeast two-hybrid experiments, where BIG1 interacted with an FKBP, and BIG2 with RIa, a regulatory subunit of cyclic AMP-activated protein kinase A (PKA). FKBP13 is a member of the FK506-binding family of immunophilins that, unlike the better known FKBP12, does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506, a widely used immunosuppressive drug, increased binding of BIG1, BIG2, and ARF to Golgi and other membranes as did a structurally related agonist. No effect was seen with a structurally related antagonist,or with cyclosporin A, or rapamycin, immunosuppressants that do not act via an FKBP. These findings are consistent with the role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through BIG1. BIG2 interaction with RIa was confirmed by co-immunoprecipitation of in vitro-translated BIG2 and Ria, as well as of the endogenous proteins from cultured Hep G2 cells. Incubation of cells with agents that raised cell cyclic AMP concentration resulted in translocation of BIG2 (and BIG1) to Golgi and other membranes. Using 28 deletion mutants of BIG2, three regions of the molecule that interacted with one or more of the four R subunits of PKA were identified. Helical wheel projections of the sequences revealed potential amphipathic helical structures characteristic of AKAPs (A kinase-anchoring proteins). The broad spectrum of signalling events controlled by cyclic AMP requires compartmentalization of PKA and its substrate(s) along with the other molecules that are involved in specific intracellular functions. These findings are consistent with a role for BIG2 in coordinating cAMP and ARF regulatory pathways.
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