Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library.
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.
Mohrlüder
Jeannine
J
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Stangler
Thomas
T
Hoffmann
Yvonne
Y
Wiesehan
Katja
K
Mataruga
Anja
A
Willbold
Dieter
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D037282:Calreticulin / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D007150:Immunohistochemistry; D008024:Ligands; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D051381:Rats; D020349:Surface Plasmon Resonance
D037282:Calreticulin; C502186:GABARAP protein, rat; D008024:Ligands; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D019151:Peptide Library