Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase.
1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.
Saxty
B A
BA
Medicine and Therapeutics (Division of Medicine), Imperial College School of Medicine, Chelsea and Westminster Hospital, London SW10 9NH.
Yadollahi-Farsani
M
M
Upton
P D
PD
Johnstone
S R
SR
MacDermot
J
J
D000818:Animals; D000077214:Becaplermin; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004318:Doxycycline / Q000494:pharmacology; D006801:Humans; D008168:Lung; D008934:Mitogens / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism
D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D011065:Poly(ADP-ribose) Polymerases; D017479:Receptors, Platelet-Derived Growth Factor; D004318:Doxycycline