P. Pomies et al., CRP1, A LIM DOMAIN PROTEIN IMPLICATED IN MUSCLE DIFFERENTIATION, INTERACTS WITH ALPHA-ACTININ, The Journal of cell biology, 139(1), 1997, pp. 157-168
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.