Calponin and the composition of smooth muscle thin filaments

Abstract

Recent work suggests that caldesmon and calponin, two actin-binding proteins found in smooth muscle, compete with each other for interaction with F-actin, implying that they may not be subunits of the same complex in vivo. Rather than associating with the same thin filaments in vivo, the two proteins actually may be localized on separate filaments possibly performing distinct functions. It is well-established that actomyosin ATPase and consequently tension in vertebrate smooth muscle are dependent on Ca2+-calmodulin-activated phosphorylation of myosin light chain subunits (reviewed in Kamm & Stull, 1985). The possibility of additional thin filament-linked modulation, raised by the pioneering work of Driska and Hartshome (1975) and Ebashi and colleagues (1977) and detailed extensively by Marston and colleagues (Marston et al., 1980; Marston & Smith, 1984), also has received much attention. This possibility became particularly attractive after Sobue and colleagues (1981) first isolated and characterized the protein caldesmon, which now is known to be localized exclusively on thin filaments (Marston & Lehman, 1985) and capable of modulating actomyosin ATPase in vitro (Sobue eta]., 1981; Ngai & Walsh, I984; Dabrowska et al., 1985; Marston & Lehman, I985; Chacko et al., I987). More recently, a second putative thin filament-linked regulatory protein, calponin, has been characterized by Takahashi and colleagues (1986, 1988, 1990), and their work on this 34-35 kDa molecule has stimulated further interest in the role played by thin filament-binding proteins in the contractility of smooth muscle. In these studies, antibody crossreactivity and molecular cloning demonstrated that regions of calponin have an amino acid sequence homologous to one of the tropomyosin binding domains of skeletal muscle troponinT, suggesting strongly that calponin is a true thin filament binding protein (Takahashi et al., 1988, 1990). Calponin also shares extensive sequence homology with the very abundant and modestly-named smooth muscle protein, SM 22, which as yet has no known function or binding affinities (Vancompernolle et al., 1990; Takahashi et al., 1990; Lees-Miller et al., 1987; Pearlstone et al., 1987). Calponin, like caldesmon, binds to actin with high affinity and also binds calmodulin (Lehman & Kaminer, 1984; Takahashi et al., 1986; Lehman, 1989; Winder & Walsh, 1990; Sutherland eta]., I990). However, with both caldesmon and calponin potentially interacting with actin, models of smooth muscle thin filaments seem to be becoming somewhat congested. Clearly a more complete understanding of the composition, assembly, and distribution of thin filaments in smooth muscle is required to clarify the roles played by these thin filament-binding proteins. A number of studies analyzing reconstituted and intact thin filaments have begun to address these questions and provide direction for future investigation. Experiments in the laboratories of Shirinsky and Dabrowska may have yielded a key piece of evidence pertinent to our understanding of how caldesmon and calponin interact with actin. In their studies, F-actin complexed with purified calponin was exposed to increasing concentrations of caldesmon; this resulted in binding of the caldesmon and displacement of bound calponin. Analogously, they also found that calponin was able to displace caldesmon bound to actin filaments (Makuch eta]., 1991). Hence, caldesmon and calponin compete for actin binding sites on thin filaments and apparently do not form a complex with each other, as do the subunits of troponin on skeletal muscle thin filaments. This would explain why, in some preparations, calponin seems to interfere with caldesmon inhibition of actomyosin ATPase (S. B. Marston, personal communication) and, in others, caldesmon with calponin inhibition (Sutherland eta]., 1990). Surprisingly, the presence of tropomyosin does not seem to influence calponin inhibition of ATPase (Makuch eta]., 1990; Winder & Walsh, 1990), and its role in calponin function and in calponin binding on thin filaments remains undefined. Even though calponin and caldesmon both bind to actin, it seems unlikely that they are randomly or even sequentially arranged on individual thin filaments in vivo. Caldesmon, at least, is regularly and periodically distributed on relatively intact, isolated, thin filaments in