Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.
ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.
Price
S R
SR
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Welsh
C F
CF
Haun
R S
RS
Stanley
S J
SJ
Moss
J
J
Vaughan
M
M
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010743:Phospholipids / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D004247:DNA; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human