Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these ‘calponin homology (CH) domains’ have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the calponin molecule. All three mammalian isoforms of calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142–163, between the CH domain and the tandem repeats, which in h1 calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 calponin are shown to harbour a second, independent actin binding motif.
- © 1998 by Company of Biologists