Identification of an FHL1 protein complex containing ACTN1, ACTN4, and PDLIM1 using affinity purifications and MS-based protein-protein interaction analysis

Mol Biosyst. 2011 Apr;7(4):1185-96. doi: 10.1039/c0mb00235f. Epub 2011 Jan 18.


Introduction: Four and a half LIM domains protein 1 (FHL1) is the most widely expressed member of the FHL family of proteins, consisting of four and a half highly conserved LIM domains. A multifunctional and integral role for FHL1 has been implicated in muscle development, structural maintenance, and signaling. To date, 27 FHL1 mutations have been identified that result in at least six different X-linked myopathies, with patients often presenting with cardiovascular complications. Since proteins assemble into dynamic complexes within the cell, FHL1 likely mediates its biological functions in conjunction with other proteins. Delineation of FHL1 interactions could provide insight into its regulatory functions.

Methods: We performed tandem affinity purification from human embryonic kidney 293 (HEK-293) cells to purify FHL1 and interacting proteins. To identify the potential interactors of FHL1 we performed a total of 9 different purifications from HEK-293 cells which included 3 experimental replicates for each biological condition: FHL1, tag control (DPYSL3), and negative control (empty vector). Purified samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Potential interactors were then verified by immunoprecipitation from mouse heart ventricles and interactions visualized in adult cardiomyocytes using 3D fluorescence microscopy.

Results: We identified a total of 310 different proteins from all 9 purifications and by applying stringent filtering criteria we eliminated all proteins found in any of the controls and only allowed those that were detected in two or more bait purification. We identified 34 high confidence potential binding partners of FHL1. We then showed that FHL1 exists as part of a complex that binds with PDLIM1, GSN and ACTN1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinin* / metabolism
  • Animals
  • Chromatography, Affinity*
  • Computational Biology
  • Gelsolin / metabolism
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins* / isolation & purification
  • Intracellular Signaling Peptides and Proteins* / metabolism
  • LIM Domain Proteins
  • Mass Spectrometry*
  • Mice
  • Microfilament Proteins* / metabolism
  • Muscle Proteins* / isolation & purification
  • Muscle Proteins* / metabolism
  • Protein Binding
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors


  • ACTN1 protein, human
  • ACTN4 protein, human
  • FHL1 protein, human
  • Gelsolin
  • Intracellular Signaling Peptides and Proteins
  • LDB2 protein, human
  • LIM Domain Proteins
  • Microfilament Proteins
  • Muscle Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Actinin