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CRP1, a LIM domain protein implicated in muscle differentiation, interacts with alpha-actinin.

Pomiès P, Louis HA, Beckerle MC - J. Cell Biol. (1997)

Bottom Line: The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells.Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton.The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.

ABSTRACT
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 alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-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-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 alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-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 alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

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Binding of [125I]α-actinin to CRP1 in a solid-phase binding assay. (A) The  specificity of the association  between CRP1 and [125I]α-actinin in a solid-phase binding assay was analyzed in a  competition experiment. A  constant amount of [125I]α-actinin (0.26 pmoles in 120  μl) was incubated in CRP1-coated wells with increasing  concentrations of unlabeled  α-actinin (+ α-actinin) or  BSA (+ BSA). In this experiment, a total of 3,076 cpm  were bound specifically to  CRP1 when no competing  unlabeled α-actinin was  added. The data are expressed as a percentage of  the maximum binding obtained when the [125I]α-actinin is incubated with the CRP1-coated wells in the absence of competing  protein. (B) From the competition experiment shown in A, the  moles of α-actinin bound to CRP1 were plotted as a function of  the free α-actinin concentration. In this particular experiment,  the α-actinin was radioiodinated to a specific activity of 5.8 × 106  cpm/μg; assuming a mol wt of 200,000 g/mol for α-actinin. The  calculated dissociation constant (Kd) was 1.9 μM. The mean dissociation constant determined from three different experiments  using two different probes is 1.8 ± 0.3 μM (mean ± SEM).
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Figure 4: Binding of [125I]α-actinin to CRP1 in a solid-phase binding assay. (A) The specificity of the association between CRP1 and [125I]α-actinin in a solid-phase binding assay was analyzed in a competition experiment. A constant amount of [125I]α-actinin (0.26 pmoles in 120 μl) was incubated in CRP1-coated wells with increasing concentrations of unlabeled α-actinin (+ α-actinin) or BSA (+ BSA). In this experiment, a total of 3,076 cpm were bound specifically to CRP1 when no competing unlabeled α-actinin was added. The data are expressed as a percentage of the maximum binding obtained when the [125I]α-actinin is incubated with the CRP1-coated wells in the absence of competing protein. (B) From the competition experiment shown in A, the moles of α-actinin bound to CRP1 were plotted as a function of the free α-actinin concentration. In this particular experiment, the α-actinin was radioiodinated to a specific activity of 5.8 × 106 cpm/μg; assuming a mol wt of 200,000 g/mol for α-actinin. The calculated dissociation constant (Kd) was 1.9 μM. The mean dissociation constant determined from three different experiments using two different probes is 1.8 ± 0.3 μM (mean ± SEM).

Mentions: The affinity of the association between α-actinin and CRP1 was characterized by a solid-phase binding assay. Purified bacterially expressed CRP1 (Fig. 3 A) was adsorbed to microtiter wells, unoccupied sites on the plastic wells were blocked with BSA, and the immobilized CRP1 was incubated with [125I]α-actinin. The amount of bound [125I]α-actinin was determined by γ counting. The specificity of the CRP1–α-actinin interaction in this solid-phase binding assay was evaluated by comparing the ability of unlabeled α-actinin or BSA to compete with radiolabeled α-actinin for binding to CRP1 (Fig. 4 A). A constant amount of [125I]α-actinin was incubated in CRP1-coated wells in the presence of increasing concentrations of competing proteins. The interaction between CRP1 and [125I]α-actinin is inhibited by the unlabeled α-actinin but not by an equivalent molar excess of BSA, demonstrating the specificity of the interaction between CRP1 and α-actinin in the solid-phase binding assay. A typical curve predicted by the simple binding reaction: CRP1 + α-A ↔ CRP1·α-A, was obtained by plotting the moles of α-actinin bound to CRP1 against the concentration of free α-actinin (Fig. 4 B). Half maximum binding in this experiment occurs at 1.9 μM free ligand. From the average of three different experiments using two different probes we calculate an average Kd of 1.8 ± 0.3 μM (mean ± SEM) for the CRP1–α-actinin interaction.


CRP1, a LIM domain protein implicated in muscle differentiation, interacts with alpha-actinin.

Pomiès P, Louis HA, Beckerle MC - J. Cell Biol. (1997)

Binding of [125I]α-actinin to CRP1 in a solid-phase binding assay. (A) The  specificity of the association  between CRP1 and [125I]α-actinin in a solid-phase binding assay was analyzed in a  competition experiment. A  constant amount of [125I]α-actinin (0.26 pmoles in 120  μl) was incubated in CRP1-coated wells with increasing  concentrations of unlabeled  α-actinin (+ α-actinin) or  BSA (+ BSA). In this experiment, a total of 3,076 cpm  were bound specifically to  CRP1 when no competing  unlabeled α-actinin was  added. The data are expressed as a percentage of  the maximum binding obtained when the [125I]α-actinin is incubated with the CRP1-coated wells in the absence of competing  protein. (B) From the competition experiment shown in A, the  moles of α-actinin bound to CRP1 were plotted as a function of  the free α-actinin concentration. In this particular experiment,  the α-actinin was radioiodinated to a specific activity of 5.8 × 106  cpm/μg; assuming a mol wt of 200,000 g/mol for α-actinin. The  calculated dissociation constant (Kd) was 1.9 μM. The mean dissociation constant determined from three different experiments  using two different probes is 1.8 ± 0.3 μM (mean ± SEM).
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Figure 4: Binding of [125I]α-actinin to CRP1 in a solid-phase binding assay. (A) The specificity of the association between CRP1 and [125I]α-actinin in a solid-phase binding assay was analyzed in a competition experiment. A constant amount of [125I]α-actinin (0.26 pmoles in 120 μl) was incubated in CRP1-coated wells with increasing concentrations of unlabeled α-actinin (+ α-actinin) or BSA (+ BSA). In this experiment, a total of 3,076 cpm were bound specifically to CRP1 when no competing unlabeled α-actinin was added. The data are expressed as a percentage of the maximum binding obtained when the [125I]α-actinin is incubated with the CRP1-coated wells in the absence of competing protein. (B) From the competition experiment shown in A, the moles of α-actinin bound to CRP1 were plotted as a function of the free α-actinin concentration. In this particular experiment, the α-actinin was radioiodinated to a specific activity of 5.8 × 106 cpm/μg; assuming a mol wt of 200,000 g/mol for α-actinin. The calculated dissociation constant (Kd) was 1.9 μM. The mean dissociation constant determined from three different experiments using two different probes is 1.8 ± 0.3 μM (mean ± SEM).
Mentions: The affinity of the association between α-actinin and CRP1 was characterized by a solid-phase binding assay. Purified bacterially expressed CRP1 (Fig. 3 A) was adsorbed to microtiter wells, unoccupied sites on the plastic wells were blocked with BSA, and the immobilized CRP1 was incubated with [125I]α-actinin. The amount of bound [125I]α-actinin was determined by γ counting. The specificity of the CRP1–α-actinin interaction in this solid-phase binding assay was evaluated by comparing the ability of unlabeled α-actinin or BSA to compete with radiolabeled α-actinin for binding to CRP1 (Fig. 4 A). A constant amount of [125I]α-actinin was incubated in CRP1-coated wells in the presence of increasing concentrations of competing proteins. The interaction between CRP1 and [125I]α-actinin is inhibited by the unlabeled α-actinin but not by an equivalent molar excess of BSA, demonstrating the specificity of the interaction between CRP1 and α-actinin in the solid-phase binding assay. A typical curve predicted by the simple binding reaction: CRP1 + α-A ↔ CRP1·α-A, was obtained by plotting the moles of α-actinin bound to CRP1 against the concentration of free α-actinin (Fig. 4 B). Half maximum binding in this experiment occurs at 1.9 μM free ligand. From the average of three different experiments using two different probes we calculate an average Kd of 1.8 ± 0.3 μM (mean ± SEM) for the CRP1–α-actinin interaction.

Bottom Line: The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells.Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton.The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.

ABSTRACT
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 alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-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-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 alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-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 alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

Show MeSH
Related in: MedlinePlus