title: Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2.
abstract: OBJECTIVE
  To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2.
  
  
  METHODS
  The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay.
  
  
  RESULTS
  The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2).
  
  
  CONCLUSIONS
  A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.
authors.affiliation: Joint Laboratory of Vascular Biology, Ruijin Hospital, Health Science Center, Shanghai, China. liqun@sibs.ac.cn
authors.forename: Qun
authors.initials: Q
authors.lastname: Li
authors.forename: Yves
authors.initials: Y
authors.lastname: Laumonnier
authors.forename: Tatiana
authors.initials: T
authors.lastname: Syrovets
authors.forename: Thomas
authors.initials: T
authors.lastname: Simmet
mesh_terms: D017306:Annexin A2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005341:Fibrinolysin / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D059748:Proteolysis; D018000:Receptors, Peptide / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D017306:Annexin A2; D018076:DNA, Complementary; D018000:Receptors, Peptide; C065181:plasmin receptor; D005341:Fibrinolysin