Glial cell line-derived neurotrophic factor-dependent recruitment of Ret into lipid rafts enhances signaling by partitioning Ret from proteasome-dependent degradation.
The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFRalphas. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.
Pierchala
Brian A
BA
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, Missouri 63110, USA. bap7@buffalo.edu
Milbrandt
Jeffrey
J
Johnson
Eugene M
EM
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D005728:Ganglia, Sympathetic / Q000166:cytology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051096:Proto-Oncogene Proteins c-ret / Q000145:classification / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
D051100:Glial Cell Line-Derived Neurotrophic Factor; D008565:Membrane Proteins; D009842:Oligopeptides; D013778:Tetrazolium Salts; D013844:Thiazoles; D025801:Ubiquitin; C106354:flotillins; D020932:Nerve Growth Factor; D051096:Proto-Oncogene Proteins c-ret; C495001:Ret protein, rat; D046988:Proteasome Endopeptidase Complex; C022616:thiazolyl blue; C078846:epoxomicin