N‐terminal domain of dystrophin

Abstract

Contro-versial experiments have been published on calmodulin binding of dystrophin. In this study, we used recombinant proteins and the techniques of affinity chromatography and ELISA to show that the N-terminal part of dystrophin binds calmodulin specifically in a calciumdependent manner. The calcium-dependent interaction of calmodulin and dystrophin does not directly regulate binding of actin to dystrophin, but may regulate dystrophin interactions with other associated proteins.

Dystrophin and utrophin: the missing links!

“…These observations may explain why Milner et al [40] were able to detect calcium binding in a GST-fusion construct comprising residues 3,107 3,400 of dystrophin, whereas Ervasti and Campbell failed to detect calcium binding in [45Ca] gel-overlays of blotted whole dystrophin [7], possibly due to the inability of whole dystrophin to re-fold correctly after blotting. Furthermore, calmodulin overlays with whole dystrophin failed to detect calmodulin binding [7] despite positive results being obtained with discrete expressed domains [13,14]. Negative results in gel-overlay experiments therefore, cannot necessarily be construed as evidence of lack of function.…”

“…Although the bacterially expressed actin binding domains from both dystrophin and utrophin bound to calmodulin affinity columns in the presence of calcium [13,14], but not in the absence, only utrophin binding to actin was inhibited by calmodulin in a calcium-dependent manner [14]. Given the high degree of sequence conservation in this region in these two proteins, the reasons for this difference in behaviour may have to await the elucidation of their tertiary structures.…”

Structure-function relationships in dystrophin and utrophin

“…One difference between the actin-binding domains of dystrophin and utrnphin is in their interaction with calmodulin. Whereas calmodulin binds to both expressed actin-binding domains in the presence of calcium but not in its absence [6,7], only the binding of utrophin to F-actin is affected by cal- cium/calmodulin [7]. In sedimentation F-actinbinding assays, half-maximum displacement of 20 pM UTR261 from 10 pM F-actin occurred at 50 pM calcium/calmodulin [7], whereas in the absence of calcium UTR261 was not displaced from F-actin even at a 10-fold molar excess of calmodulin.…”

Human mineralocorticoid receptor interacts with actin under mineralocorticoid ligand modulation

“…F-actin was stored at 4°C in buffer A (2 mM Tris-HC1 pH 8.0, 10 mM KC1, 0.1 mM CaC12 and 1 mM NAN3). Preparation of G-actin was previously described [14]. F and G-actin were titrated by spectrophotometric measurement in a Kontron Uvikon 930 using actin e 1% at 280 nm = 1.1.…”