Molecular interactions of N-RAP, a nebulin-related protein of striated muscle myotendon junctions and intercalated disks.
N-RAP is a recently discovered muscle-specific protein that is concentrated at the myotendon junctions in skeletal muscle and at the intercalated disks in cardiac muscle. The C-terminal half of N-RAP contains a region with sequence homology to nebulin, while a LIM domain is found at its N-terminus. N-RAP is hypothesized to perform an anchoring function, linking the terminal actin filaments of myofibrils to protein complexes located beneath the sarcolemma. We used a solid-phase assay to screen myofibrillar and junctional proteins for binding to several recombinant fragments of N-RAP, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal half including the LIM domain (N-RAP-NH), and the region of N-RAP between the super repeat region and the LIM domain (N-RAP-IB). Actin is the only myofibrillar protein tested that exhibits specific binding to N-RAP, with high-affinity binding to N-RAP super repeats, and 10-fold weaker binding to N-RAP-IB. In contrast, myosin, isolated myosin heads, tropomyosin, and troponin exhibited no specific interaction with N-RAP domains. A recombinant fragment corresponding to the C-terminal one-fourth of vinculin also binds specifically to N-RAP super repeats, while no specific N-RAP binding activity was observed for other regions of the vinculin molecule. Finally, talin binds with high affinity to the LIM domain of N-RAP. These results support our hypothesis that N-RAP is part of a complex of proteins that anchors the terminal actin filaments of the myofibril to the membrane, and functions in transmitting tension from the myofibrils to the extracellular matrix.
D000199:Actins / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000378:metabolism; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D009994:Osmolar Concentration; D012508:Sarcolemma / Q000378:metabolism / Q000502:physiology; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism