Department of Cell & Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3G5.
Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, ON, Canada M5S 3B2.
Proc Natl Acad Sci U S A. 2017 Jul 11;114(28):7385-7390. doi: 10.1073/pnas.1705765114. Epub 2017 Jun 22.
High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.
高海拔环境通过降低氧气、压力和温度相对于低海拔生境，对生物提出了一系列生化和生理挑战。在多种陆地温血动物中，已经确定了蛋白质水平对低氧高海拔条件的适应；然而，在鱼类等水生变温动物中，类似的适应并没有得到广泛的描述。在酶蛋白中，通过稳定性和活性之间的功能权衡来实现冷适应，这通常是通过活性部位之外的取代来介导的。对于信号蛋白（例如 G 蛋白偶联受体 (GPCR)）是否会对低温表现出自然变异，人们知之甚少。视紫红质 (RH1) 是介导弱光视觉的温度敏感视觉色素，为在模型 GPCR 中增强对热适应的理解提供了机会。在这里，我们研究了跨越一系列海拔高度的安第斯山脉鲶鱼系统中视紫红质功能的进化。使用分子进化分析和定点诱变实验，我们提供了 RH1 冷适应的证据。我们发现，在高海拔鲶鱼中，正选择作用下的独特氨基酸取代发生在 RH1 分子内氢键网络的两端。通过诱变引入这些位点的天然高海拔变体对光谱调谐的影响有限，但会降低黑暗状态和光激活视紫红质的稳定性，加速配体结合形式的衰减。与冷适应酶一样，这种表型可能补偿了低温诱导的动力学速率降低——介导杆状敏感性和视觉性能的视紫红质特性。我们的结果支持自然变异在增强 GPCR 对低温的性能中的作用。
