Ubiquitination Of GluN2B Subunit-containing NMDA Receptors By Cbl-b In The Spinal Cord Dorsal Horn

Objective: N-methyl-D-aspartic acid(NMDA)subtype glutamate receptors(NMDAR)are tetrameric heterooligomers that are composed of combinations of GluN1,GluN2 and/or GluN3 subunits.Early after birth,the excitatory synapses predominantly express GluN2 B subunit-containing NMDAR.With the development,the contents of synaptic GluN2 B gradually decline,which are replaced by GluN2 A.The mechanisms underlying the switch of synaptic NMDAR subunit composition are largely unknown as yet.Ubiquitination is one of the post-translational protein modifications that participate in the regulation of a series of cellular functions.Casitas B-lineage lymphoma b(Cbl-b)is one of the widely distributed E3 ubiquitin ligases.This study was designed to investigate the regulatory effects of Cbl-b on GluN2 B and NMDAR subunit switch.Methods: Immunohistochemistry was used to examine the distribution of Cbl-b in spinal cord dorsal horn.By performing co-immunoprecipitation and western blot,we studied the role of Cbl-b in the modification of GluN2 B ubiquitination and synaptic expression.We cultured spinal neurons in vitro and investigated the effect of Cbl-b on surface GluN2 B expression by using immunocytochemistry.Results:(1)Cbl-b was detectable in spinal cord dorsal horn neurons rather than spinal microglia or astrocytes.(2)The immunocytochemical experiments showed that Cbl-b was distributed in the spinal excitatory synapses,which might be involved in the synapse modification.(3)Cbl-b specifically ubiquitinated NMDA receptor GluN2 B subunit,an effect that was not observed with C-Cbl,the Cbl-b homology.(4)Cbl-b was unable to ubiquitinate NMDA receptor GluN2 A subunit,AMPA(?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid–subtype glutamate receptors)receptor GluA1 and GluA2 subunits.(5)Cbl-b conjugated GluN2 B with Lys63-linked ubiquitin rather than Lys48-linked ubiquitin.(6)Cbl-b-mediated ubiquitination decreased the expression of GluN2 B on plasma membrane.(7)From birth to adulthood,the ubiquitination level of GluN2 B gradually increased in spinal cord dorsal horn of mice and then decreased.(8)When ubiquitinated by Cbl-b,GluN2 B was removed away from synapses,which gradually decreased the expression of GluN2 B at excitatory synapses with development.(9)The sh RNA knockdown of Cbl-b inhibited NMDAR subunit switch during development.Conclusions: The ubiquitin modification by Cbl-b reduced synaptic GluN2 B contents and contributed to the developmental NMDAR subunit switch.