Ubiquitination Precedes Internalization and Proteolytic Cleavage of Plasma Membrane-bound Glycine Receptors

Abstract

The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric ␣1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, ␣1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.The efficiency of synaptic transmission depends critically on a dense packing of neurotransmitter receptors in the postsynaptic membrane. At fast synapses, ligand-gated ion channels (LGICs) 1 mediate the postsynaptic response. Different lines of evidence indicate that the distribution and density of LGICs in the plasma membrane are regulated tightly. In differentiating muscle fibers, the formation of a densely packed postsynaptic matrix of nicotinic acetylcholine receptors (nAChRs) at the developing motor endplate requires restriction of gene expression to subsynaptic nuclei, efficient internalization and degradation of extrasynaptic receptors, synaptic clustering by rapsyn, and slowing of the turnover of synaptically accumulated nAChRs (1, 2). Inversely, muscle denervation (2) or blockade of neurotransmission by ␣-bungarotoxin (3) causes a loss of nAChRs from the postsynaptic membrane because of an increased rate of protein turnover (4). Because nAChR degradation occurs only after internalization and lysosomal targeting of the receptor protein (4, 5

Multifunctional Basic Motif in the Glycine Receptor Intracellular Domain Induces Subunit-specific Sorting

“…The proteolytic cleavage probably occurred between the conserved regions C1 and C3 of the TM3-4 loop. This localization is consistent with a study reporting ubiquitination and subsequent cleavage of a C-terminal-tagged GlyR construct expressed in Xenopus oocytes (32). The T7-tagged fragment we observed, however, neither harbored the basic motif 346 …”

Incompatibility between a Pair of Residues from the Pre-M1 Linker and Cys-loop Blocks Surface Expression of the Glycine Receptor

“…The underlying mechanism is that this mutation misfolds the protein, thus prolongs association with chaperon proteins and in consequence restrains surface expression (41). Moreover, the ER quality control machinery coupled with the ubiquitinproteasome system has been shown to regulate surface expression of the GlyR and nAChR (42)(43)(44).…”

The ubiquitin-proteasome pathway and synaptic plasticity

“…Endocytosis of other neurotransmitter receptors might be regulated by ubiquitination. A glycine receptor has been shown to be internalized upon ubiquitination (Buttner et al 2001). A protein associated with GABA A receptors, Plic-1, indirectly controls the removal of GABA A through endocytosis (Bedford et al 2001).…”