title: The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis.
abstract: BACKGROUND
  During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain.
  
  
  RESULTS
  Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation.
  
  
  CONCLUSIONS
  As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.
authors.forename: Xiudan
authors.initials: X
authors.lastname: Zheng
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. kliao@sibs.ac.cn.
authors.forename: Kan
authors.initials: K
authors.lastname: Liao
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010712:Phosphatidic Acids / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D059748:Proteolysis; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D010712:Phosphatidic Acids; D021382:Protein Sorting Signals; C433810:SH3KBP1 protein, human; D050721:Proto-Oncogene Proteins c-cbl; C512478:EGFR protein, human; D066246:ErbB Receptors