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Abstract：
A major unsolved question in vertebrate photoreceptor biology is the mechanism of rhodopsin transport to the outer segment. In rhodopsin-like class A G protein-coupled receptors, hydrophobic interactions between C-terminal α-helix 8 (H8), and transmembrane α-helix-1 (TM1) have been shown to be important for transport to the plasma membrane, however whether this interaction is important for rhodopsin transport to ciliary rod outer segments is not known. We examined the crystal structures of vertebrate rhodopsins and class A G protein-coupled receptors and found a conserved network of predicted hydrophobic interactions. In Xenopus rhodopsin (xRho), this interaction corresponds to F313, L317, and L321 in H8 and M57, V61, and L68 in TM1. To evaluate the role of H8-TM1 hydrophobic interactions in rhodopsin transport, we expressed xRho-EGFP where hydrophobic residues were mutated in Xenopus rods and evaluated the efficiency of outer segment enrichment. We found that substituting L317 and M57 with hydrophilic residues had the strongest impact on xRho mislocalization. Substituting hydrophilic amino acids at positions L68, F313, and L321 also had a significant impact. Replacing L317 with M resulted in significant mislocalization, indicating that the hydrophobic interaction between residues 317 and 57 is exquisitely sensitive. The corresponding experiment in bovine rhodopsin expressed in HEK293 cells had a similar effect, showing that the H8-TM1 hydrophobic network is essential for rhodopsin transport in mammalian species. Thus, for the first time, we show that a hydrophobic interaction between H8 and TM1 is critical for efficient rhodopsin transport to the vertebrate photoreceptor ciliary outer segment.
摘要：
脊椎动物光感受器生物学中一个尚未解决的主要问题是视紫红质转运到外节的机制。在视紫红质样A类G蛋白偶联受体中，C端α-螺旋8(H8)之间的疏水相互作用，和跨膜α-螺旋-1（TM1）已被证明是重要的运输到质膜，然而，这种相互作用对于视紫红质运输到睫状杆外段是否很重要尚不清楚。我们检查了脊椎动物视紫红质和A类G蛋白偶联受体的晶体结构，并发现了预测的疏水相互作用的保守网络。在非洲爪狼视紫红质（xRho）中，这种相互作用对应于H8中的F313、L317和L321以及TM1中的M57、V61和L68。为了评估H8-TM1疏水相互作用在视紫红质转运中的作用，我们表达了xRho-EGFP，其中疏水残基在非洲爪狼棒中突变，并评估了外段富集的效率。我们发现用亲水残基取代L317和M57对xRho错位具有最强的影响。在位置L68、F313和L321处取代亲水性氨基酸也具有显著影响。用M代替L317会导致严重的误定位，这表明残基317和57之间的疏水相互作用非常敏感。相应实验在牛视紫红质HEK293细胞中的表达也有类似的效果，表明H8-TM1疏水网络对于哺乳动物物种中的视紫红质转运至关重要。因此，第一次,我们表明，H8和TM1之间的疏水相互作用对于将视紫红质有效转运到脊椎动物感光睫状外段至关重要。
