Measurement of epidermal growth factor receptor turnover and effects of Cdc42.
Ligand-induced degradation represents an essential component of the overall regulation of EGF receptor (EGFR)-coupled signal transduction. Following activation, EGFRs are monoubiquitinated, subsequently sorted by ubiquitin-interaction-based sorting machinery, and transported to multivesicular bodies (MVBs) and lysosomes for degradation. The Rho-family small G-protein, Cdc42, has been implicated in the regulation of EGFR degradation. Here we describe routine methods for assaying EGFR endocytosis and degradation. In addition, we have introduced procedures for determining the effects of Cdc42 and its downstream targets, in particular, ACK (Activated Cdc42-associated Kinase) and p85Cool-1 (Cloned out of library)/Pix (for Pak-interactive exchange factor), on EGFR degradation.
Lin
Qiong
Q
Weis Center for Research, Geisinger Clinic, Danville, PA, USA.
Yang
Wannian
W
Cerione
Richard A
RA
D000818:Animals; D018797:Cell Cycle Proteins / Q000494:pharmacology; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism* / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D020662:Guanine Nucleotide Exchange Factors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000528:radiation effects*; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D020764:cdc42 GTP-Binding Protein