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Characterization of the actin binding site on smooth muscle filamin
M C Lebart1, C Méjean, D Casanova
1Centre National de la Recherche Scientifique, U 249 Institut National de la Santé et de la Recherche Médicale, Ecole Pratique des Hautes Etudes, Université de Montpellier I, France.
Abstract:
We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.
Insights
Researchers identified a specific filamin protein sequence (121-147) crucial for binding to filamentous actin. This binding involves hydrophobic interactions, suggesting a distinct interface from alpha-actinin.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Filamin is a cytoskeletal protein known to interact with filamentous actin.
- Alpha-actinin also binds actin, with a conserved 27-mer sequence implicated in this interaction.
Purpose of the Study:
- To identify the specific region of filamin responsible for binding to filamentous actin.
- To compare the actin-binding mechanism of filamin with that of alpha-actinin.
Main Methods:
- Digestion of filamin with Staphylococus aureus V8 protease to isolate an NH2-terminal fragment.
- Cosedimentation assays to assess the interaction between the filamin fragment and filamentous actin.
- Enzyme-linked immunosorbent assay (ELISA) and synthetic peptide approaches using anti-peptide antibodies.
Main Results:
- An NH2-terminal filamin fragment (M(r) = 70,000) was isolated and shown to bind filamentous actin.
- Evidence suggests the filamin sequence 121-147, homologous to alpha-actinin's actin-binding site, is key for actin interaction.
- The hydrophobic region (141-147) of this peptide is significantly involved in actin binding.
- Filamin-actin interaction shows different ionic strength dependence compared to alpha-actinin, indicating distinct binding interfaces.
Conclusions:
- The filamin sequence 121-147 plays a major role in its interaction with filamentous actin.
- Both hydrophobic and potentially hydrophilic regions of filamin contribute to actin binding, differentiating it from alpha-actinin's mechanism.