Structural determinants of alpha4beta2 nicotinic acetylcholine receptor trafficking.
The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the alpha4beta2 AChR subunits were required for cell surface expression of alpha4beta2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the alpha4 AChR subunit attenuates cell surface expression of mutant alpha4beta2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the beta2 AChR subunit abolishes cell surface expression of mutant alpha4beta2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the beta2 AChR subunit only poorly inhibits trafficking of mutant alpha4beta2 AChR to the cell surface. All mutant alpha4beta2 AChRs exhibit high-affinity binding for [3H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type alpha4beta2 AChRs colocalize with the Golgi marker giantin, whereas mutant alpha4beta2 AChRs fail to do so. The striking difference between mutant alpha4 versus mutant beta2 AChR subunits on cell surface expression of mutant alpha4beta2 AChRs points to a cooperative or regulatory role for the alpha4 AChR subunit and an obligatory role for the beta2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of alpha4beta2 AChR ER export.
Ren
Xiao-Qin
XQ
Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Cheng
Shi-Bin
SB
Treuil
Magdalen W
MW
Mukherjee
Jayanta
J
Rao
Jayaraman
J
Braunewell
K H
KH
Lindstrom
Jon M
JM
Anand
Rene
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007668:Kidney; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011725:Pyridines / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D019086:Bridged Bicyclo Compounds, Heterocyclic; D011725:Pyridines; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C403531:nicotinic receptor alpha4beta2; D007930:Leucine; C082748:epibatidine