Abstract
The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.
Highlights
A recent report by Marston et al (1994) suggested that there exists a second CaM-binding site in the C-terminal region of CaD, in addition to the previously identified CaM-binding site (Zhan et al, 1991), and that site B alone is involved in the CaM sensitivity of CaD's inhibitory action
Their viewpoint was mainly supported by two findings: (a) a recombinant fragment (H9) of CaD that lacks site A, but contains site B, shows Ca 2 +/CaM-dependent regulation of acto-S1 ATPase similar to that of intact CaD; (b) a synthetic peptide (M73), which contains site A and binds CaM but does not inhibit the acto-S1 ATPase activity, does not compete with intact CaD in the presence of CaM, and there is no re-inhibition
We have confirmed that the peptide stretch encompassing site B binds CaM; but we found that site A is involved in the CaD-CaM interaction that is responsible for the reversal of CaD's inhibitory action of the actomyosin ATPase activity
Summary
Vol 270, No 34, Issue of August 25, pp. 19964-19968, 1995 Printed in U.S.A. Identification of the Functionally Relevant Calmodulin Binding Site in Smooth Muscle Caldesmon*. The GSl7C-induced activation of muscle contraction is most likely a result of direct competition for actin, rather than for CaM, between endogenous CaD and the added peptide This in tum, strongly supports the idea that in vivo CaD plays an inhibitory role in the regulation of smooth muscle contraction, and CaM apparently neutralizes such inhibition by interacting with CaD at the GS17C sequence. Marston et al (1994) found that a site A-containing peptide (M73, from Ser-657 to Gly-670) did bind CaM, it could not compete with CaD for CaM and did not restore the inhibition of actomyosin ATPase activity, whereas a site B-containing fragment H2 (from Thr-626 to Leu-TlO) did From these results, they concluded that it is site B, not site A, that is functionally relevant for CaD's action (Marston et al, 1994).
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