Abstract
Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.
Highlights
Heavy chain isoforms, nonmuscle myosin heavy chains (NMHC)1 II-A, II-B, and II-C, which are the products of three different genes termed MYH9 [4, 5], MYH10 [5], and MYH14 [6, 7] in humans, respectively
We introduced the mutation R722C, which is homologous to R709C in NMHC II-B and R702C in NMHC II-A, into both the inserted (R730C) and noninserted (R722C) NMHC II-C isoforms
Whereas deletion of myosin II-A leads to defects in the visceral endoderm and cell-cell adhesion in the embryo, resulting in lethality as early as E6.5 [8], loss of myosin II-B allows development to progress to E14.5, with death being accompanied by abnormalities in the heart and brain [16, 30]
Summary
Heavy chain isoforms, nonmuscle myosin heavy chains (NMHC) II-A, II-B, and II-C, which are the products of three different genes termed MYH9 [4, 5], MYH10 [5], and MYH14 [6, 7] in humans, respectively. The R726S mutation in NMHC II-C at an amino acid homologous to the R702C mutation in NMHC II-A results in an autosomal dominant hearing loss in humans [14]. After we initiated this work, a report appeared describing humans with an autosomal dominant hearing loss who had a mutation in NMHC II-C at R726S [14]. This is homologous to Arg-722 (or Arg-730 in the inserted isoform) in mice, so we expressed murine-inserted HMM II-C that had the R730S mutation
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