GTP-BINDING PROTEIN STRUCTURE/FUNCTION STUDIES

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ADP-ribosylation factors (ARFs), a highly conserved family of about 20 kDa-guanine nucleotide-binding proteins that stimulate cholera toxin (CT) activity in vitro, are well known regulators of specific vesicular trafficking events. ARF with GTP bound is in an active conformation and associates with membranes which may facilitate its interaction with one of several possible effectors, such as the GTP-binding protein-activated isoform of phospholipase D (PLD). In an effort to identify domains of ARF responsible for its interaction with effectors, we generated amino- terminally truncated ARFs, chimeric ARFs and point mutants. Amino- terminal truncation of ARF1 significantly reduced its ability to activate PLD, but not its ability to bind GTP. Chimeric proteins, derived from ARF1 and human ARF-like protein (ARL1), a protein that causes minimal activation of PLD, were previously used to establish that amino acids 1 to 73 of ARF are involved in PLD activation and that amino acids 74-181 are involved in CT activation. The chimeric construct ARF28ARL (the amino-terminal 28 amino acids of ARF1 plus the carboxy-terminal 153 amino acids of ARL1) had less ability to activate PLD than did an ARF50ARL construct. Thus, residues between amino acids 28 and 50 that differ in ARF and ARL, are involved in the activation of PLD and it appears that at least two distinct regions of ARF are involved in the activation of PLD. Recently, the interaction of ARF with the heterotrimeric G-protein beta/gamma subunits was shown to play a role in endosome fusion, consistent with cross-talk between these major regulatory proteins. To test the possibility that ARF interacts with CT via a sequence similar to the beta/gamma-interaction region, which is present in beta-adrenergic receptor kinase (betaARK), but not in rhodopsin kinase (RK), we utilized fusion protein constructs of betaARK, RK, and ARF, as well as peptides with the putative interaction sequence. GST-betaARK and betaARK-derived peptides inhibited ARF stimulation of CT, whereas GST-RK did not. Moreover, a fusion protein corresponding to the C-terminal half of ARF also inhibited ARF stimulation of CT. Based on these and prior studies with ARF-ARL chimeric proteins, it appears that the sequence in ARF that is similar to the beta/gamma-interaction site of betaARK can affect cholera toxin - ARF interactions.