Interaction of the GTP-binding and GTPase-activating Domains of ARD1 Involves the Effector Region of the ADP-ribosylation Factor Domain

Abstract

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins and members of the Ras superfamily, originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin and more recently recognized as critical participants in vesicular trafficking pathways and phospholipase D activation. ARD1 is a 64-kDa protein with an 18-kDa carboxyl-terminal ARF domain (p3) and a 46-kDa amino-terminal extension (p5) that is widely expressed in mammalian tissues. Using recombinant proteins, we showed that p5, the amino-terminal domain of ARD1, stimulates the GTPase activity of p3, the ARF domain, and appears to be the GTPase-activating protein (GAP) component of this bifunctional protein, whereas in other members of the Ras superfamily a separate GAP molecule interacts with the effector region of the GTP-binding protein. p5 stimulated the GTPase activity of p3 but not of ARF1, which differs from p3 in several amino acids in the effector domain. After substitution of 7 amino acids from p3 in the appropriate position in ARF1, the chimeric protein ARF1(39-45p3) bound to p5, which increased its GTPase activity. Specifically, after Gly40 and Thr45 in the putative effector domain of ARF1 were replaced with the equivalent Asp and Pro, respectively, from p3, functional interaction of the chimeric ARF1 with p5 was increased. Thus, Asp25 and Pro30 of the ARF domain (p3) of ARD1 are involved in its functional and physical interaction with the GTPase-activating (p5) domain of ARD1. After deletion of the amino-terminal 15 amino acids from ARF1(39-45p3), its interaction with p5 was essentially equivalent to that of p3, suggesting that the amino terminus of ARF1(39-45p3) may interfere with binding to p5. These results are consistent with the conclusion that the GAP domain of ARD1 interacts with the effector region of the ARF domain and thereby stimulates GTP hydrolysis.