A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells.
Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.
Vazhappilly
Rema
R
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore.
Wee
Karen Siaw-Ling
KS
Sucher
Nikolaus J
NJ
Low
Chian-Ming
CM
D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers; D003720:Densitometry; D004587:Electrophoresis, Agar Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000502:physiology; D018995:Myosin Heavy Chains / Q000378:metabolism; D024462:Myosin Type II / Q000502:physiology*; D016716:PC12 Cells; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000378:metabolism
D017931:DNA Primers; C473649:NR1A NMDA receptor, rat; D016194:Receptors, N-Methyl-D-Aspartate; D025801:Ubiquitin; D012313:RNA; D046988:Proteasome Endopeptidase Complex; D024462:Myosin Type II; D018995:Myosin Heavy Chains