tailieunhanh - Báo cáo khoa học: Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABAA) receptor-associated protein (GABA-RAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABAAreceptors, and belongs to a family of proteins involved in intra-cellular vesicular transport processes, such as autophagy and intra-Golgi transport. | ỊFEBS Journal Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library Jeannine Mohrluder1 2 Thomas Stangler1 2 Yvonne Hoffmann1 2 Katja Wiesehan2 Anja Mataruga3 and Dieter Willbold1 2 1 Institut fur Physikalische Biologie Heinrich-Heine-Universitat Dusseldorf Germany 2 Institut fur Neurowissenschaften und Biophysik INB-2 Molekulare Biophysik Forschungszentrum Julich Germany 3 Institut fur Neurowissenschaften und Biophysik INB-1 Zellulare Biophysik Forschungszentrum Julich Germany Keywords calreticulin GABAa receptor GABARAP phage display screening protein-protein interaction Correspondence T. Stangler or D. Willbold INB-2 Molekulare Biophysik Forschungszentrum Julich 52425 Juulich Germany Fax 49 2461 61 8766 Tel 49 2461 61 2100 E-mail stangler@ or Received 29 June 2007 revised 30 July 2007 accepted 29 August 2007 doi 4-Aminobutyrate type A GABAa receptor-associated protein GABA-RAP is a ubiquitin-like modifier implicated in the intracellular trafficking of GABAa 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 Ca2 -dependent chaperone and a Ca2 -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 calreticu-lin as a potential GABARAP binding protein. GABARAP binding to cal-reticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant Kd 64