Twitchin from Molluscan Catch Muscle

Abstract

The phosphorylation state of the myosin thick filament-associated mini-titin, twitchin, regulates catch force maintenance in molluscan smooth muscle. The full-length cDNA for twitchin from the anterior byssus retractor muscle of the mussel Mytilus was obtained using PCR and 5rapid amplification of cDNA ends, and its derived amino acid sequence showed a large molecule (ϳ530 kDa) with a motif arrangement as follows: (Ig) 11 (IgFn 2 ) 2 Ig(Fn) 3 Ig(Fn) 2 Ig(Fn) 3 (Ig) 2 (Fn) 2 (Ig) 2 FnKinase(Ig) 4 . Other regions of note include a 79-residue sequence between Ig domains 6 and 7 (from the N terminus) in which more than 60% of the residues are Pro, Glu, Val, or Lys and between the 7th and 8th Ig domains, a DFRXXL motif similar to that thought to be necessary for high affinity binding of myosin light chain kinase to F-actin. Two major phosphorylation sites, i.e. D1 and D2, were located in linker regions between Ig domains 7 and 8 and Ig domains 21 and 22, respectively. Correlation of the phosphorylation state of twitchin, using antibodies specific to D1 and D2, with mechanical properties suggested that phosphorylation of both D1 and D2 is required for relaxation from the catch state.Some molluscan smooth muscles exhibit a mechanical state known as "catch" (1, 2), which is characterized by long term force maintenance with a very low energy requirement (3-6). Catch muscles such as the anterior byssus retractor muscle (ABRM) 1 of the mussel Mytilus are innervated by cholinergic and serotonergic nerves. Cholinergic excitation gives rise to an increase in intracellular [Ca 2ϩ ] and force output, but force is maintained in the catch state despite a subsequent decrease in [Ca 2ϩ ] to near-resting levels (7). Serotonergic nerve activation results in rapid relaxation of catch force without any change in intracellular [Ca 2ϩ ] (7). The relaxation is mediated by an increase in cAMP and activation of PKA (8, 9).The central role of PKA in relaxation of catch force led to a series of experiments to identify the protein whose phosphorylation state has such a dramatic effect on regulation of force maintenance in this muscle. Only one protein, having a molecular mass of ϳ600 kDa, showed a change in phosphorylation that corresponded to the release of catch force in permeabilized ABRM (10). Based on a partial cDNA of the purified isolated protein, it was found to be a homologue of the mini-titin, twitchin (11), that is associated with the thick filament (12). The mechanism by which twitchin controls force output seems to reside in the ability of unphosphorylated twitchin to slow the detachment of calcium-free myosin from actin, primarily by slowing the rate of ADP release from attached cross-bridges (13,14). In the presence of phosphorylated twitchin, crossbridge detachment is fast at low intracellular [Ca 2ϩ ] (14). In vitro studies on both native and reconstituted thick filaments from Mytilus also show the primary role that twitchin and its phosphorylation state play in regulation of the catch state (15).Under in vitro conditi...

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

“…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 N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments

“…In order to test the effect of phosphorylation of the recombinant proteins on the binding to the thin filament, proteins were phosphorylated by the addition of the catalytic subunit of cyclic AMP-dependent protein kinase (Calbiochem) (150 kilounits/ml) for 1 h at 20°C. Phosphorylation was confirmed using Pro Q Diamond phosphoprotein stain (Invitrogen), and the D1 phosphorylation-dependent antibody (12) confirmed nearly 100% phosphorylation of the D1 site.…”

“…Twitchin is a mini-titin that is composed of Ig and fibronectin domains as well as a kinase domain near the C terminus (11). The domain organization of twitchin from Mytilus (12) is shown in Fig. 1.…”

“…In vitro, twitchin is phosphorylated by PKA with a stoichiometry of about 3 mol of phosphate/mol of twitchin (15). The D1 phosphorylation site is located between the 7th and 8th Ig domain in the N-terminal part of the molecule, and the D2 site is near the C terminus between the 21st and 22nd Ig domain (12,15). Another site of phosphorylation shares a seven-amino acid sequence with the D1 site, is located in the same Ig linker region, and is referred to as DX (16).…”

“…Peptides ranging from 47 to 52 amino acids included the following: PEVK1, DX, D1, and D2. PEVK1 is from a PEVK-rich region of twitchin that is shorter and has a lower frequency of PEVK residues than titin from vertebrate muscle (12). This peptide was chosen because the PEVK domain from titin has been shown to interact with actin (28 -30).…”