title: Processing of ADP-ribosylated integrin alpha 7 in skeletal muscle myotubes.
abstract: Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol-anchored, arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta 1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434, USA.
authors.forename: A
authors.initials: A
authors.lastname: Zolkiewska
authors.forename: J
authors.initials: J
authors.lastname: Moss
mesh_terms: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD; D002478:Cells, Cultured; D039001:Integrin alpha Chains; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology
chemical_list: D015703:Antigens, CD; D039001:Integrin alpha Chains; D016023:Integrins; C085725:integrin alpha7; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases