Ren Xiao-Qin, Cheng Shi-Bin, Treuil Magdalen W, Mukherjee Jayanta, Rao Jayaraman, Braunewell K H, Lindstrom Jon M, Anand Rene
Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Neurosci. 2005 Jul 13;25(28):6676-86. doi: 10.1523/JNEUROSCI.1079-05.2005.
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.
烟碱型乙酰胆碱受体（AChR）转运的结构决定因素尚未完全阐明。疏水残基存在于对内质网（ER）输出或内吞转运很重要的短基序中。因此，我们测试了α4β2 AChR亚基胞质结构域中高度保守的疏水残基（主要是亮氨酸）对于α4β2 AChRs细胞表面表达是否必需。α4 AChR亚基中F350、L351、L357和L358突变为丙氨酸会减弱突变型α4β2 AChRs的细胞表面表达。β2 AChR亚基中同源位置的F342、L343、L349和L350突变为丙氨酸会消除突变型α4β2 AChRs的细胞表面表达。亮氨酸残基的疏水性是其功能的主要决定因素，因为β2 AChR亚基中L343突变为另一种疏水氨基酸苯丙氨酸只会微弱地抑制突变型α4β2 AChR向细胞表面的转运。所有突变型α4β2 AChRs对[3H]依博加因均表现出高亲和力结合。在tsA201细胞和分化的SH-SY5Y神经细胞中，野生型α4β2 AChRs与高尔基体标记物巨蛋白共定位，而突变型α4β2 AChRs则不然。突变型α4与突变型β2 AChR亚基在突变型α4β2 AChRs细胞表面表达上的显著差异表明，α4 AChR亚基在ER输出中起协同或调节作用，而β2 AChR亚基起必需作用。总体而言，我们的结果首次确定了AChR亚基中对于α4β2 AChR ER输出至关重要的结构决定残基。
