Adjacent basic amino acid residues recognized by the COP I complex and ubiquitination govern endoplasmic reticulum to cell surface trafficking of the nicotinic acetylcholine receptor alpha-Subunit.
The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of alpha-gamma-alpha-delta-beta. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg(313)-Lys(314) sequence in the large cytoplasmic loop of the alpha-subunit to K314Q promotes the trafficking of the mutant unassembled alpha-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q alpha-subunit with gamma-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type alpha-subunits when co-expressed with the beta-, delta-, and gamma-subunits. This suggests that the Arg(313)-Lys(314) sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the alpha-subunit. Although unassembled K314Q alpha-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q alpha-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface.
Department of Pharmacology, University of California, San Diego, La Jolla, Califronia 92093, USA.
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Keller
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Lindstrom
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Ellisman
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Taylor
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D020755:Coat Protein Complex I / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D011978:Receptors, Nicotinic / Q000378:metabolism*; D015398:Signal Transduction; D014452:Ubiquitins / Q000378:metabolism
D020755:Coat Protein Complex I; D011978:Receptors, Nicotinic; D014452:Ubiquitins