Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is ∼800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH₂-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca²⁺/calmodulin, as well as G protein–coupled signal transduction in the sarcomere.

The sarcomeric Z‐disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by α‐actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of α‐actinin into the Z‐disk. Titin interacts via a single binding site with the two central spectrin‐like repeats of the outermost pair of α‐actinin molecules. In the central Z‐disk, titin can interact with multiple α‐actinin molecules via their C‐terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and α‐actinin in vitro, and are expected to constrain the path of titin in the Z‐disk. In thick skeletal muscle Z‐disks, titin filaments cross over the Z‐disk centre by ∼30 nm, suggesting that their α‐actinin‐binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z‐disk, where overlapping titin filaments and their interactions with the α‐actinin rod and C‐terminal domain can account for the essential ultrastructural features.