Pydi Sai Prasad, Singh Nisha, Upadhyaya Jasbir, Bhullar Rajinder Pal, Chelikani Prashen
Department of Oral Biology, University of Manitoba, The Manitoba Institute of Child Health, Winnipeg, MB R3E 0W4, Canada.
Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):231-6. doi: 10.1016/j.bbamem.2013.08.009. Epub 2013 Aug 29.
Bitter taste receptors (T2Rs) belong to the superfamily of G protein-coupled receptors (GPCRs). T2Rs are chemosensory receptors with important therapeutic potential. In humans, bitter taste is perceived by 25 T2Rs, which are distinct from the well-studied Class A GPCRs. The activation mechanism of T2Rs is poorly understood and none of the structure-function studies are focused on the role of the important third intracellular loop (ICL3). T2Rs have a unique signature sequence at the cytoplasmic end of fifth transmembrane helix (TM5), a highly conserved LxxSL motif. Here, we pursue an alanine scan mutagenesis of the ICL3 of T2R4 and characterize the functionality of 23 alanine mutants. We identify four mutants, H214A, Q216A, V234A and M237A, that exhibit constitutive activity. To our surprise, the H214A mutant showed very high constitutive activity over wild type T2R4. Interestingly, His214 is highly conserved (96%) in T2Rs and is present two amino acids below the LxxSL motif in TM5. Molecular modeling shows a dynamic network of interactions involving residues in TM5-ICL3-TM6 that restrain the movement of the helices. Changes in this network, as in the case of H214A, Q216A, V234A and M237A mutants, cause the receptor to adopt an active conformation. The conserved LxxSL motif in TM5 performs both structural and functional roles in this process. These results provide insight into the activation mechanism of T2Rs, and emphasize the unique functional role of ICL3 even within the GPCR subfamilies.
苦味受体（T2Rs）属于G蛋白偶联受体（GPCRs）超家族。T2Rs是具有重要治疗潜力的化学感应受体。在人类中，25种T2Rs可感知苦味，它们与已得到充分研究的A类GPCRs不同。T2Rs的激活机制尚不清楚，且没有任何结构-功能研究聚焦于重要的第三细胞内环（ICL3）的作用。T2Rs在第五跨膜螺旋（TM5）的胞质端有一个独特的特征序列，即高度保守的LxxSL基序。在此，我们对T2R4的ICL3进行丙氨酸扫描诱变，并对23个丙氨酸突变体的功能进行表征。我们鉴定出四个表现出组成型活性的突变体，即H214A、Q216A、V234A和M237A。令我们惊讶的是，H214A突变体相对于野生型T2R4表现出非常高的组成型活性。有趣的是，His214在T2Rs中高度保守（96%），且位于TM5中LxxSL基序下方两个氨基酸处。分子模拟显示了一个涉及TM5-ICL3-TM6中残基的动态相互作用网络，该网络限制了螺旋的运动。如H214A、Q216A、V234A和M237A突变体的情况一样，该网络的变化会导致受体采取活性构象。TM5中保守的LxxSL基序在此过程中发挥结构和功能作用。这些结果为T2Rs的激活机制提供了见解，并强调了ICL3即使在GPCR亚家族中也具有独特的功能作用。
