ABSTRACT
FHL1, FHL2, and FHL3 are members of the four and one-half LIM domain protein subclass that are expressed in striated muscles. Here we show that FHL2 and FHL3 are novel alpha(7)beta(1) integrin-interacting proteins. They bind both the alpha- and the beta-subunit as well as different splice isoforms. The minimal binding sites for FHL2 and FHL3 on beta(1A)-chain overlap, whereas on alpha(7A) and alpha(7B) subunits they are situated adjacent. Determining the binding sites for integrins on FHL2 or FHL3 revealed that the suprastructure of the whole molecule is important for these associations, rather than any single LIM domain. Immunofluorescence studies with cells expressing full-length FHL proteins or their deletion mutants showed that FHL2 and FHL3 but not FHL1 colocalize with integrins at cell adhesion sites. Further, their recruitment to the membrane results from binding to either the alpha- or the beta-chain of the integrin receptor. The association of FHL2 or FHL3 with integrin receptors neither influences attachment of cells to different substrates nor changes their migration capacity. However, in cardiac and skeletal muscles, FHL2 and FHL3, respectively, are colocalized with alpha(7)beta(1) integrin receptor at the periphery of Z-discs, suggesting a role in mechanical stabilization of muscle cells.
Subject(s)
Antigens, CD/chemistry , Homeodomain Proteins/chemistry , Integrin alpha Chains/chemistry , Integrin beta1/chemistry , Muscle Proteins , Muscles/metabolism , Transcription Factors/chemistry , Actins/chemistry , Alternative Splicing , Amino Acid Sequence , Animals , Binding Sites , Cell Adhesion , Cell Line , Cell Movement , Cells, Cultured , Cytoplasm/metabolism , Cytoskeletal Proteins/chemistry , DNA/chemistry , Dystroglycans , Extracellular Matrix/metabolism , Focal Adhesions , Glutathione Transferase/metabolism , Humans , Immunoblotting , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , LIM-Homeodomain Proteins , Membrane Glycoproteins/chemistry , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscles/cytology , Mutation , NIH 3T3 Cells , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Time Factors , Two-Hybrid System TechniquesABSTRACT
FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.