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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 α1 GlyRs generated inXenopus 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, α1 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.
P2X1 receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X1receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn284 remains unused because of a proline in the +4 position. The four other sites (Asn153, Asn184, Asn210, and Asn300) carryN-glycans, but solely Asn300 located only eight residues upstream of the predicted reentry loop of P2X1acquires complex-type carbohydrates. Like parent P2X1, glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn153 or Asn210 diminishes or increases functional expression levels, respectively. In addition, elimination of Asn210 causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X1 receptors is severely impaired or abolished, respectively. We conclude that at least oneN-glycan per subunit of either position is absolutely required for the formation of P2X1 receptors and that individual N-glycans possess marked positional effects on expression levels (Asn154, Asn210) and ATP potency (Asn210).
