Mouse RIC-3, an endoplasmic reticulum chaperone, promotes assembly of the alpha7 acetylcholine receptor through a cytoplasmic coiled-coil domain.
RIC-3 (resistant to inhibitor of cholinesterase) is a transmembrane protein, found in invertebrates and vertebrates, that modulates the surface expression of a variety of nicotinic acetylcholine receptors (nAChRs) in neurons and other cells. To understand its mechanism of action, we investigated the cellular location, transmembrane topology and cellular mechanism by which RIC-3 facilitates alpha7 assembly and surface expression in cultured mammalian cells. We show that the mouse protein is targeted to the ER by the first 31 aa which act as a cleavable signal sequence. The mature protein is a single-pass type I transmembrane protein whose N terminus resides in the lumen of the ER with the coiled-coil domain in the cytoplasm. RIC-3, which binds both unfolded and folded alpha7 subunits, facilitates the surface expression of receptor principally by promoting the folding and assembly of the alpha7 subunits in the ER into fully polymerized receptor. Functional analysis shows that facilitation of surface expression of alpha7 in mammalian cells is reduced in RIC-3 mutants lacking the signal peptide, the lumenal segment or the coiled-coil domain, but not in mutants lacking the long C-terminal region downstream of the coiled-coil domain. We show that the coiled-coil domain of mRIC-3 is not required for the interaction of mRIC-3 with alpha7, but does mediate a homotypic interaction between molecules of mRIC-3. We suggest that efficient assembly of the homomeric alpha7 nAChR may thus require mRIC-3 self-association through the cytoplasmic coiled-coil domain and suggest a model by which this may occur.
Wang
Ying
Y
University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA. yingw17@usc.edu
Yao
Yun
Y
Tang
Xiao-Qing
XQ
Wang
Zuo-Zhong
ZZ
D000595:Amino Acid Sequence; D000818:Animals; D004721:Endoplasmic Reticulum / Q000235:genetics*; D006031:Glycosylation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000502:physiology; D021381:Protein Transport; D011978:Receptors, Nicotinic / Q000096:biosynthesis*; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579166:Chrna7 protein, mouse; D008565:Membrane Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D011978:Receptors, Nicotinic; C544672:Ric-3 protein, mouse; D064569:alpha7 Nicotinic Acetylcholine Receptor