The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization.
By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.
Jeanclos
E M
EM
Neuroscience Center of Excellence and Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Lin
L
L
Treuil
M W
MW
Rao
J
J
DeCoster
M A
MA
Anand
R
R
D048948:14-3-3 Proteins; D000109:Acetylcholine / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000378:metabolism; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D014981:Xenopus
D048948:14-3-3 Proteins; D000109:Acetylcholine; D000409:Alanine; D005576:Colforsin; D000242:Cyclic AMP; D018076:DNA, Complementary; D007074:Immunoglobulin G; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase