J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/10/157/12 $2.00
Volume 139, Number 1, October 6, 1997 157-168

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with -Actinin

Pascal Pomiès,^* Heather A. Louis,^* and Mary C. Beckerle^*

^* Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is -actinin. We have shown that the CRP1--actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The K_d for the CRP1--actinin interaction is 1.8 ± 0.3 µM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and -actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that -actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of -actinin. In reciprocal mapping studies, we showed that -actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH₂-terminal 107 amino acids (aa) of CRP1. To determine whether the -actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH₂-terminal part of CRP1 that contains the -actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with -actinin may be critical for its role in muscle differentiation.

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