The NH2 terminus of titin spans the Z-disc: Its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity

Abstract. Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3–4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH 2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for �-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH 2-terminal overlap and number of �-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH 2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament–regulated sarcomeric assembly. Key words: titin • �-actinin • Z-disc • titin-cap (T-cap) • sarcomer