The α7 Integrin as a Target Protein for Cell Surface Mono-ADP-Ribosylation in Muscle Cells

Abstract

A membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle and its gene was isolated from a skeletal muscle cDNA library. The enzyme was a glycosylphosphatidyl-inositol-linked protein, present on the surface of differentiated skeletal muscle myoblasts (myotubes). Following incubation of cultured, intact myotubes with [adenylate-³²P]NAD and analysis by SDS-PAGE, a major radiolabeled protein of 97/140 kDa (reduced/ nonreduced conditions) was observed. It was identified as integrin a 7 based on its size, binding to a laminin affinity column, immunoprecipitation with a monoclonal antibody, and partial amino acid sequencing.

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, whereas the transferase is attached via a GPI-anchor to the cell surface, the processing of ADP-ribosylated integrin a7 was investigated. ³²P label was rapidly removed from [32P]ADP-ribosylated integrin a7, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5’-monophosphate, alternative substrates for 5′-nucleotide phosphodiesterase. The processed integrin a7 was not susceptible to subsequent ADP-ribosylation, although the amount of surface integrin a7 remained constant. During the processing, no loss of label was observed from integrin a7 radiolabeled with [¹⁴C]NAD, containing ¹⁴C in the nicotinamide-proximal ribose, consistent with a 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 the presently known ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.