CRP1, A LIM DOMAIN PROTEIN IMPLICATED IN MUSCLE DIFFERENTIATION, INTERACTS WITH ALPHA-ACTININ

Members of the cysteine-rich protein (CRP) family are LIM domain prote ins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to c haracterize the repertoire of CRP binding partners. In order to identi fy proteins that interact with CRP1, a prominent protein in fibroblast s and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound t o the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actini n. We have shown that the CRP1-alpha-actinin interaction is direct, sp ecific, and saturable in both solution and solid-phase binding assays. The K-d for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 mu M. T he results of the in vitro protein binding studies are supported by do uble-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-ac tinin coimmunoprecipitates with CRP1 from a detergent extract of smoot h muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-act inin. In reciprocal mapping studies, we showed that alpha-actinin inte racts with CRP1-LIM1, a deletion fragment that contains the NH2-termin al 107 amino acids (aa) of CRP1. To determine whether the alpha-actini n binding domain of CRP1 would localize to the actin cytoskeleton in l iving cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1 (aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjec ted into cells. By indirect immunofluorescence, we have determined tha t full-length CRP1 and CRP1-LIM1 localize along the actin stress fiber s whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collecti vely these data demonstrate that the NH2-terminal part of CRP1 that co ntains the alpha-actinin-binding site is sufficient to localize CRP1 t o the actin cytoskeleton. The association of CRP1 with alpha-actinin m ay be critical for its role in muscle differentiation.