Ubiquitination precedes internalization and proteolytic cleavage of plasmamembrane-bound glycine receptors

The inhibitory glycine receptor (GlyR) in developing spinal neurones is int ernalized efficiently upon antagonist inhibition. Here we used surface labe ling combined with affinity purification to show that homopentameric alpha1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragmen ts of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the en docytotic pathway. Consistent with this interpretation, elevation of the ly sosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cle avage. Prior to internalization, al GlyRs are conjugated extensively with u biquitin in the plasma membrane. Our results are consistent with ubiquitina tion regulating the endocytosis and subsequent proteolysis of GlyRs residin g in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a cru cial role in synaptic plasticity by determining postsynaptic receptor numbe rs.