TrkA receptor endolysosomal degradation is both ubiquitin and proteasome dependent.
Gaps in our knowledge exist regarding the degradation of the tropomyosin-regulated kinase A (TrkA) receptor after addition of neurotrophin, nerve growth factor (NGF). TrkA is rapidly and transiently ubiquitinated upon addition of NGF. Here, we demonstrate that the polyubiquitin tag plays a definitive role in receptor sorting. Treatment of PC12 cells with lactacystin prevented NGF-dependent deubiquitination and degradation of TrkA. However, treatment with methylamine, bafilomycin or leupeptin, did not prevent NGF-dependent deubiquitination but blocked the degradation of TrkA. Employing co-immunoprecipitation, biochemical fractionation and confocal microscopy, the kinetics of receptor trafficking post-internalization was observed to occur as a sequel from endosome/multivesicular body, proteasomes, culminating with degradation in the lysosomes. The trafficking of the polyubiquitin-deficient TrkA receptor mutant K485R was impaired and likewise failed to degrade revealing that the receptor escapes degradation. The interaction of TrkA with proteasomes was confirmed by purification and co-immunoprecipitation. We provide evidence that proteasomal deubiquitinating enzymes trim K63-ubiquitin chains from the TrkA receptor prior to its delivery to lysosomes for degradation. Taken together, our results reveal the existence of a novel proteasome-dependent step in receptor degradation.
Geetha
Thangiah
T
Department of Biological Sciences, Program in Cellular and Molecular Biosciences, Auburn University, Auburn, AL 36849, USA.
Wooten
Marie W
MW
D000818:Animals; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000737:chemistry; D008247:Lysosomes / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008744:Methylamines / Q000737:chemistry; D009154:Mutation; D016716:PC12 Cells; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D051381:Rats; D020917:Receptor, trkA / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry*
D007976:Leupeptins; D018942:Macrolides; D008744:Methylamines; D025801:Ubiquitin; C057620:bafilomycin A; C027451:methylamine; D020917:Receptor, trkA; D046988:Proteasome Endopeptidase Complex; C032854:leupeptin