Identification of calreticulin as ligand of GABARAP by phage display screening of a peptide library

Mohrlüder, J.; Hoffmann, Y.; Wiesehan, K.; Mataruga, A.; Stangler, T.; Willbold, D.

doi:10.1111/j.1742-4658.2007.06073.x

Journal Article

Identification of calreticulin as ligand of GABARAP by phage display screening of a peptide library

Mohrlüder, J.FZJ* ; Stangler, T.FZJ* ; Wiesehan, K.FZJ* ; Hoffmann, Y.FZJ* ; Mataruga, A.FZJ* ; Willbold, D.FZJ*

2007
Wiley-Blackwell Oxford [u.a.]

The FEBS journal 274(21), 5543 - 5555 (2007) [10.1111/j.1742-4658.2007.06073.x]

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Please use a persistent id in citations: http://hdl.handle.net/2128/4688 http://hdl.handle.net/2128/4687 doi:10.1111/j.1742-4658.2007.06073.x

Abstract: 4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.

Keyword(s): Amino Acid Motifs (MeSH) ; Amino Acid Sequence (MeSH) ; Animals (MeSH) ; Binding Sites (MeSH) ; Calreticulin: chemistry (MeSH) ; Calreticulin: metabolism (MeSH) ; Cells, Cultured (MeSH) ; Immunohistochemistry (MeSH) ; Ligands (MeSH) ; Microtubule-Associated Proteins: chemistry (MeSH) ; Microtubule-Associated Proteins: metabolism (MeSH) ; Models, Molecular (MeSH) ; Molecular Sequence Data (MeSH) ; Peptide Fragments: chemistry (MeSH) ; Peptide Fragments: metabolism (MeSH) ; Peptide Library (MeSH) ; Rats (MeSH) ; Surface Plasmon Resonance (MeSH) ; Calreticulin ; GABARAP protein, rat ; Ligands ; Microtubule-Associated Proteins ; Peptide Fragments ; Peptide Library ; J ; calreticulin (auto) ; GABA(A) receptor (auto) ; GABARAP (auto) ; phage display screening (auto) ; protein-protein interaction (auto)