Abstract
Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (
K
d
=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.