EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate.
EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.
Department of Pharmacology and the Holden Comprehensive Cancer Center, The Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Kathryn A
KA
Stern
Trenton L
TL
Place
Nancy L
NL
Lill
D066258:Amphiregulin; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D015536:Down-Regulation; D066255:EGF Family of Proteins; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000819:agonists / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
C587848:AREG protein, human; D066258:Amphiregulin; D017931:DNA Primers; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D025801:Ubiquitin; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors; C067419:CBL protein, human