title: UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.
abstract: Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.
authors.affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Khosrow
authors.initials: K
authors.lastname: Rezvani
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Teng
authors.forename: Yaping
authors.initials: Y
authors.lastname: Pan
authors.forename: John A
authors.initials: JA
authors.lastname: Dani
authors.forename: Jon
authors.initials: J
authors.lastname: Lindstrom
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: García Gras
authors.forename: J Michael
authors.initials: JM
authors.lastname: McIntosh
authors.forename: Mariella
authors.initials: M
authors.lastname: De Biasi
mesh_terms: D000818:Animals; D019921:Biotinylation; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004622:Embryo, Mammalian; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000648:ultrastructure*; D018408:Patch-Clamp Techniques; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology
chemical_list: D007931:Leucyl Aminopeptidase; D008565:Membrane Proteins; D046988:Proteasome Endopeptidase Complex; D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D014452:Ubiquitins; C515819:UBXN4 protein, human; C541434:Ubxn2a protein, rat