In Vitro Mutational and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily.
In Vitro Mutational and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily.
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DOI：
10.1371/journal.pone.0141712
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Feinstein P
中科院分区：
文献类型：
--
作者：
Bubnell J;Jamet S;Tomoiaga D;D'Hulst C;Krampis K;Feinstein P
我们对典型小鼠气味受体（OR） M71进行了广泛的突变分析，以确定在异源表达系统中抑制质膜运输的ORs的特性。我们利用M71::GFP融合蛋白直接评估了M71在体外异源细胞或体内嗅感觉神经元（OSNs）中的质膜定位和功能。M71::GFP的OSN表达与未标记的M71相比，活性差异很小。然而，M71::GFP即使在辅助蛋白RTP1S或m - β 2ar存在的情况下也不能迁移到质膜上。为了了解OR是否包含内部“杀伤序列”，我们突变了约15个在非OR GPCR转运超家族中未发现的高度保守的OR特异性氨基酸；这些变种人都没有拯救人口贩卖。添加不同的氨基末端信号序列或不同的糖基化基序都不能产生转运。在高度保守的GPCR基序NPxxY中添加m - β 2ar的氨基和羧基末端结构域或突变Y289A并不能挽救质膜运输。靶向诱变在异源细胞中挽救质膜定位的失败表明，OR转运缺陷可能不是由于保守的共线基序，而是由于OR家族的整体氨基酸组成。因此，我们进行了一个比较OR和其他胺受体超家族的计算机分析。我们发现，ORs含有更少的带电残基和更多的疏水残基分布在整个蛋白质和保守的整体氨基酸组成。根据我们的分析，我们推测，如果不进行显著的氨基酸修饰，可能很难在体外将高水平的ORs运输到细胞表面。最后，我们观察到蛋氨酸和组氨酸残基的特异性增加以及色氨酸残基的显著减少，这表明这些变化为嗅觉神经元提供了它们发挥作用所需的特殊特征。
We performed an extensive mutational analysis of the canonical mouse odorant receptor (OR) M71 to determine the properties of ORs that inhibit plasma membrane trafficking in heterologous expression systems. We employed the use of the M71::GFP fusion protein to directly assess plasma membrane localization and functionality of M71 in heterologous cells in vitro or in olfactory sensory neurons (OSNs) in vivo. OSN expression of M71::GFP show only small differences in activity compared to untagged M71. However, M71::GFP could not traffic to the plasma membrane even in the presence of proposed accessory proteins RTP1S or mβ2AR. To ask if ORs contain an internal “kill sequence”, we mutated ~15 of the most highly conserved OR specific amino acids not found amongst the trafficking non-OR GPCR superfamily; none of these mutants rescued trafficking. Addition of various amino terminal signal sequences or different glycosylation motifs all failed to produce trafficking. The addition of the amino and carboxy terminal domains of mβ2AR or the mutation Y289A in the highly conserved GPCR motif NPxxY does not rescue plasma membrane trafficking. The failure of targeted mutagenesis on rescuing plasma membrane localization in heterologous cells suggests that OR trafficking deficits may not be attributable to conserved collinear motifs, but rather the overall amino acid composition of the OR family. Thus, we performed an in silico analysis comparing the OR and other amine receptor superfamilies. We find that ORs contain fewer charged residues and more hydrophobic residues distributed throughout the protein and a conserved overall amino acid composition. From our analysis, we surmise that it may be difficult to traffic ORs at high levels to the cell surface in vitro, without making significant amino acid modifications. Finally, we observed specific increases in methionine and histidine residues as well as a marked decrease in tryptophan residues, suggesting that these changes provide ORs with special characteristics needed for them to function in olfactory neurons.