title: Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.
abstract: Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.
authors: Department of Molecular Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, 10691 Stockholm, Sweden.
authors: Erik
authors: E
authors: Vernet
authors: Emma
authors: E
authors: Lundberg
authors: Mikaela
authors: M
authors: Friedman
authors: Nicolò
authors: N
authors: Rigamonti
authors: Sandra
authors: S
authors: Klausing
authors: Per-Ake
authors: PA
authors: Nygren
authors: Torbjörn
authors: T
authors: Gräslund
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D066246:ErbB Receptors