Abstract
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.
Highlights
Identification of the GTPase-activating proteins (GAPs) Domain of ARD1—Incubation of p3 or p5 with affinity-purified polyclonal antibodies raised against recombinant p3 or p5, respectively, markedly reduced, in a concentration-dependent manner, the ability of p5 to stimulate hydrolysis of GTP bound to p3 (Fig. 1), whereas the antibodies did not affect GTP binding. 30 g of either antibody completely blocked p5-stimulated GTPase activity (Fig. 1), whereas up to 50 g of an anti-GST antibody had no effect
These results indicated that anti-p3 or anti-p5 antibodies inhibited GTP hydrolysis more effectively when the two domains of ARD1 were present in separate proteins than when covalently linked in recombinant ARD1
The affinity-purified carboxyl-terminal antibody only slightly reduced (ϳ20%) the amount of GTP bound to p3, perhaps by affecting the structure of the GTP binding pocket of the ADP-ribosylation factors (ARFs) domain. 30 g of carboxylterminal antibody reduced GTP hydrolysis by about 25% (Fig. 1), suggesting that when antibody was bound to p3, the affinity between the two domains of ARD1 was reduced, or the rate of GTP hydrolysis was decreased directly. 30 g of the affinitypurified amino-terminal antibody affected neither GTP binding nor GTP hydrolysis (Fig. 1), suggesting that the amino terminus of p5 might not be involved in the GAP activity
Summary
Vol 273, No 5, Issue of January 30, pp. 2553–2560, 1998 Printed in U.S.A. Molecular Characterization of the GTPase-activating Domain of ADP-ribosylation Factor Domain Protein 1 (ARD1)*. We had reported that the ARF domain of ARD1 binds GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. Using ARD1 truncations, it appears that amino acids 101–190 are critical for GAP activity, whereas residues 190 –333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. A relatively new class of GAPs for heterotrimeric G proteins includes the RGS (regulator of G protein signaling) family [19] They can contribute to desensitization induced by a prolonged signal or act as long term attenuators of signal amplitude, presumably by stimulating GTP hydrolysis (for review, see Ref. 20). By site-specific mutagenesis, we demonstrate further that in p5 an intact zinc finger motif, two arginines, and a sequence that resembles a consensus motif present in Rho/Rac GAPs are required for GAP activity
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