Structure/function analysis of the CLR motif, a novel and conserved actin-binding motif in vertebrates and invertebrates

Research project P 14285 Structure function analysis of the CLR motif Mario GIMONA 08.05.2000 The smooth muscle protein calponin exhibits two distinct domains: an N-terminal calponin homology (CH) domain and a C-terminal set of three 29 residue tandem repeats (Calponin-like repeats or CLRs). These two domain types define two new, partially overlapping families of cytoskeletal and signaling proteins. The present project concerns itself with the elucidation of the role of the tandem CLR motifs, identified in proteins of vertebrates, invertebrates and yeast and in up to seven copies in nematodes (the body wall muscle proteins UNC 87 and OV9M from C.elegans and O. volvulus, respectively). We have recently shown that the isolated tandem repeats of calponin can associate with actin stress fibers raising the possibility that UNC 87 may also bind to actin via the CLR motif. A comprehensive database analysis of all proteins containing either single or multiple CLRs suggests that this motif may represent an archetypal actin-binding module. We now aim at defining the precise mode of interaction between the CLR motif and filamentous actin and whether or not co-filament proteins, including tropomyosin influence binding. Experiments using live cells will be carried out to investigate the effects of CLR motifs on the stability and turnover of actin stress fibers. Finally, we aim at determining the three dimensional structure of the CLR motifs in calponin and UNC 87 so as to reveal the molecular interface with the actin filament. The results of this study should further our understanding of the structure and function of this novel protein module and its significance for the organization of the actin cytoskeleton. Since sequences related to the CLR motif are thought to serve as primary antigens in humans upon infection by various parasites expressing CaP-related proteins, the results of this study will have impact not only on the field of actomyosin regulation, but also on aspects of parasitology and immunology.

The goal of this research project was to determine the molecular basis for the interaction of the CLIK 23 protein module with the actomyosin system. We have demonstrated for the first time that members of the calponin (CaP) family perform opposing roles in the stabilization and regulation of the actin cytoskeleton in vivo. We identified UNC-87 as an actin bundling protein and highlighted that the CLIK 23 repeats represent archetypal actin filament- binding modules conserved from yeast to man. We could also show that actin fibers decorated with h1CaP remain stable while SM22 decorated actin bundles were rapidly reorganized into podosomes in response to PDBu. Our results demonstrated the involvement of CaP and SM22 in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We further provided evidence for the existence of two functionally distinct actin filament populations and introduced a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK 23 repeats. This work breaks new grounds in terms of regulation and remodelling of the smooth muscle cytoskeleton. We demonstrated for the first time that smooth muscle cells have the intrinsic potential to form podosomes, and that the CLIK 23 repeats are sufficient to suppress their formation in cultured cells. We have thus formulated the hypothesis that i) CLIK 23 repeats contribute "conformational" stability to actin filaments, and that ii) the stabilization of only a subpopulation of actin filaments by the CLIK 23 modules reflects structural differences within actin filaments, likely driven by the binding of the actin-interacting component(s). Considering that phorbol ester-induced formation of podosomes is also observed in human intestinal smooth muscle cells (HISM) this particular cytoskeletal remodelling may be a general feature of smooth muscle cells, which is tightly regulated in normal tissue by balanced levels of CaP and SM22 and driven by the CLIK 23 modules. Reducing the endogenous levels of CaP would thus favor the formation of podosomes and amplify the potential for cell migration, causing an increased invasive behaviour of dedifferentiated smooth muscle cells in malignant vascular anomalies. We have further established the rat A7r5 vascular smooth muscle system as a twofold unique model system, namely for i) studying smooth muscle-specific functions and interactions, and ii) for the investigation of podosome formation in vivo.