COMPLETE CDNA SEQUENCE AND TISSUE LOCALIZATION OF N-RAP, A NOVEL NEBULIN-RELATED PROTEIN OF STRIATED-MUSCLE

We have cloned and sequenced the full-length cDNA of N-RAP, a novel ne bulin-related protein, from mouse skeletal muscle. The N-RAP message i s specifically expressed in skeletal and cardiac muscle, but is not de tected by Northern blot in non-muscle tissues. The full-length N-RAP c DNA contains an open reading frame of 3,525 base pairs which is predic ted to encode a protein of 133 kDa. A 587 amino acid region near the C -terminus is 45 % identical to the actin binding region of human nebul in, containing more than 2 complete 245 residue nebulin super repeats. The N-terminus contains the consensus sequence of a cysteine-rich LIM domain, which may function in mediating protein-protein interactions. These data suggest that the encoded protein may link actin filaments to some other proteins or structure. We expressed full-length N-RAP in Escherichia coli, as well as the nebulin-like super repeat region of N-RAP (N-RAP-SR) and the region between the LIM domain and N-RAP-SR (N -RAP-IB). An anti-N-RAP antibody raised against a 30 amino acid peptid e corresponding to sequence from N-RAP-IB detected recombinant N-RAP a nd N-RAP-IB, but failed to detect N-RAP-SR. This antibody specifically identified a 185 kDa band as N-RAP on immunoblots of mouse skeletal a nd cardiac muscle proteins. In an assay of actin binding to electropho resed and blotted proteins, we detected significant actin binding to e xpressed nebulin super repeats and N-RAP-SR, but only a trace amount o f binding to N-RAP-IB. In immunofluorescence experiments, N-RAP was fo und to be localized at the myotendinous junction in mouse skeletal mus cle and at the intercalated disc in cardiac muscle. Based on its domai n organization, actin binding properties, and tissue localization, we propose that N-RAP play role in anchoring the terminal actin filaments in the myofibril to the membrane and may be important in transmitting tension from the myofibrils to the extracellular matrix. (C) 1997 Wil ey-Liss, Inc.