Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains

Cell Motil Cytoskeleton. 1999;44(1):1-22. doi: 10.1002/(SICI)1097-0169(199909)44:1<1::AID-CM1>3.0.CO;2-8.

Abstract

Nebulette, a 107 kDa protein associated with the I-Z-I complex of cardiac myofibrils, may play an important role in the assembly of the Z-line. Determination of the complete primary structure of 1011 residue human fetal nebulette reveals a four-domain layout similar to skeletal muscle nebulin: a short N-terminal domain, followed by 22 nebulin-like repeats that are linked to a C-terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for nebulette in the Z-line, the complete coding sequence or fusions of nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z-lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP-repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP-repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z-lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z-lines in cardiomyocytes. Supporting the cellular localization work, recombinant nebulette fragments bind to actin, tropomyosin, and alpha-actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z-lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z-lines, suggesting that the ligands for their interactions are specific to these muscular substructures.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Carrier Proteins
  • Cells, Cultured
  • Chick Embryo
  • Cytoskeletal Proteins
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • Gene Expression
  • Gene Library
  • Green Fluorescent Proteins
  • Humans
  • LIM Domain Proteins
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Muscle Proteins / genetics*
  • Muscle Proteins / physiology
  • Muscle, Skeletal / metabolism
  • Myocardium / cytology
  • Myocardium / metabolism
  • Myofibrils / metabolism
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repetitive Sequences, Nucleic Acid*
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • src Homology Domains*

Substances

  • Actins
  • Carrier Proteins
  • Cytoskeletal Proteins
  • DNA, Complementary
  • LIM Domain Proteins
  • Luminescent Proteins
  • Muscle Proteins
  • NEBL protein, human
  • Recombinant Fusion Proteins
  • nebulin
  • Green Fluorescent Proteins

Associated data

  • GENBANK/AF047368