title: Determining the role of Rab7 in constitutive and ligand-mediated epidermal growth factor receptor endocytic trafficking using single cell assays.
abstract: RAB proteins are essential for the proper membrane trafficking of a number of proteins. Each of the 60+ RABs that have been identified has a discrete role in coordinating the movement from one subcellular compartment to another. Early attempts at deciphering the roles of individual RAB proteins relied heavily on the use of activating and/or dominant negative mutants (Ceresa, Histol Histopathol 21:987-993, 2006). However, overexpression of mutant proteins can lead to misleading information; high levels of expression can drive low affinity (and possibly, nonphysiological) interactions as well as cause mislocalization. The use of RNAi for transient protein knock down will reveal which membrane trafficking steps absolutely require the attenuated RAB. When determining the role of RAB protein in epidermal growth factor receptor (EGFR) membrane trafficking, there are special considerations. The EGFR undergoes constitutive and ligand-mediated endocytic trafficking. Both affect receptor signaling, but via different mechanisms. Here, we discuss how to experimentally dissect those two processes.
authors: Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, 40202, USA, brian.ceresa@louisville.edu.
authors: Brian P
authors: BP
authors: Ceresa
mesh_terms: D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
chemical_list: D008024:Ligands; D034741:RNA, Small Interfering; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D066246:ErbB Receptors; D020691:rab GTP-Binding Proteins