MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

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ADP-ribosylation factors constitute a family of 20-Kda guanine nucleotide-binding proteins, which stimulate in a GTP-dependent manner, cholera toxin-catalyzed ADP-ribosylation of Gs`, proteins unrelated to Gs` and simple guanidino compounds as well as the toxin A1 protein. ARFs are evolutionarily well conserved and present in all eukaryotes from Giardia to mammals, and participate in intracellular protein trafficking. Six mammalian ARFs, two yeast ARFs and two Giardia ARFs have been cloned. All of these ARFs, synthesized in E. coli, enhanced cholera toxin ADP- ribosyltransferaes in the presence of GTP and phospholipid/detergent. ARFs share ~65-96% amino acid identity and contain several consensus sequences. In general, differences in amino acid sequence are concentrated near the amino-terminal regions and the carboxyl halves. It was reported that the amino-terminal region of ARF is critical for membrane targeting, interaction with lipid, and ARF activity. A newly recognized gene of the ARF superfamily was isolated from HL60, and human fetal and rat brain CDNA libraries, which encodes a 64-Kda guanine nucleotide-binding protein containing an 18-Kda, functional ARF domain at its carboxyl terminus, termed ARD1 (for ARF domain). ARD1 MRNAS of 3.7 and 4.1 kb were detected in all rat tissues and in MRNAS derived from mouse, rabbit, and human tissues. ARD1 is highly conserved between rat and human. The ARF domain of ARD1 contains sequences believed to be involved in guanine nucleotide-binding and GTP hydrolysis (phosphate binding). Recombinant ARD1 or the ARF domain of ARD1, which lacks the 15 amino acid corresponding to the amino-terminal region of ARFs stimulated, in a GTP-dependent manner, cholera toxin ADP- ribosyltransferase activity. These results support the conclusion that the amino-terminal region of ARF proteins is not required for activation of cholera toxin and that the characteristic features of ARF proteins may occur as domains of larger mammalian proteins.