Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini, respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25·nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.

Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM) 2 and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch. Catch muscle develops active tension following an increase of the intracellular [Ca 2ϩ ] induced by secretion of acetylcholine. Myosin is activated by direct binding of Ca 2ϩ to the regulatory myosin light chain and initiates a relative sliding between thick and thin filaments (1). After a decrease of intracellular [Ca 2ϩ ] to resting levels, the catch state is formed where tension is maintained over long periods of time with little energy consumption (2, 3). Catch tension is abolished by secretion of serotonin and an increase of intracellular [cAMP] with the resulting activation of cAMP-dependent protein kinase and phosphorylation of twitchin (4, 5). Twitchin phosphorylation is required for relaxation of the muscle from catch. For this cycle to repeat, dephosphorylation of twitchin is necessary (6). Thus, in this scheme, twitchin is a major regulator of the catch state.Molluscan twitchin is known as a myosin-binding protein belonging to the titin/connectin superfamily. It is a single polypeptide of 530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin type 3-like motifs in addition to a single kinase domain homologous to the catalytic domain of myosin light chain kinase of vertebrate smooth muscle (7). There are several possible phosphorylation sites in molluscan twitchin recognized by cAMP-dependent protein kinase, and two, D1 and D2, have been identified. The D1 phosphorylation site (Ser-1075) is in the linker region between the 7th and 8th Ig motifs (numbering from the N terminus). The D2 site (Ser-4316) is in the linker region between the 21st and 22nd Ig motifs. Additional sites are found close to D1, but are thought not to be vital for catch regulation.The molecular mechanisms underlying development and maintenance of the catch state have been controversial for several years. One theory proposes that catch reflected attached frozen or slowly cycling cross-bridges (8, 9). What distinguished the attached cross-bridge from the detached relaxed state is not clear. Also it was suggested that interactions between thick filaments, other than cross-bridges, or between thin and thick filaments are responsible for the catch contraction (10). In either of the latter cases, the cross-bridge (myosin head) was not involved.Recently we found that a twitchin fragment including the D2 phosphorylation site and its flanking Ig motifs (TWD2-S) interacted with myosin and actin in a phosphorylation-sensitive manner, and it was suggested that this trimeric complex contributed to tension maintenance in catch (11). TWD2-S bound to a region of the actin molecule known also to interact with loop 2 of myosin that is involved in the ATP-driven movement of myosin with actin (12). In the present study, we show that the myosin loop 2 binds to TWD2-S using competitive cosedimentation assays and isothermal titration calorimetry (ITC). Th...