Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits
Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits
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DOI：
10.1074/jbc.ra118.006173
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发表时间：
2019-03-29
影响因子：
4.8
通讯作者：
Procko, Erik
中科院分区：
文献类型：
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作者：
Park, Jihye;Selvam, Balaji;Procko, Erik
C类G蛋白偶联受体（GPCR）是一种专性二聚体，对神经元对内源性和环境刺激的反应特别重要。通过大的胞外结构域的配体识别导致跨膜区域的重组以激活G蛋白信号传导。虽然单个结构域的结构是已知的，但C类GPCR的完整架构和受体活化期间结构域间偶联的机制尚不清楚。通过筛选人C类甜味受体亚基T1 R2的诱变文库，我们增强了表面表达，并确定了一个二元细胞内保留基序，其调节表面表达和与其异源二聚体伴侣T1 R3的共运输。使用高度表达的T1 R2变体，通过在人细胞中与T1 R3共运输的全面突变扫描来鉴定沿着所有结构域内的整个亚基的二聚化位点沿着。数据进一步揭示，细胞外富含半胱氨酸的结构域的C末端需要适当折叠以用于T1 R3二聚化和共运输，但不用于单独的T1 R2的表面表达。这些结果指导了活细胞中T1 R2-T1 R3二聚体的建模，其预测了围绕中心轴的结构域的扭曲排列，以及跨膜结构域环和富含半胱氨酸的结构域之间的连续折叠结构。这些见解对C类GPCR中结构域之间的构象变化如何耦合具有影响。
Class C G protein-coupled receptors (GPCRs) are obligatory dimers that are particularly important for neuronal responses to endogenous and environmental stimuli. Ligand recognition through large extracellular domains leads to the reorganization of transmembrane regions to activate G protein signaling. Although structures of individual domains are known, the complete architecture of a class C GPCR and the mechanism of interdomain coupling during receptor activation are unclear. By screening a mutagenesis library of the human class C sweet taste receptor subunit T1R2, we enhanced surface expression and identified a dibasic intracellular retention motif that modulates surface expression and co-trafficking with its heterodimeric partner T1R3. Using a highly expressed T1R2 variant, dimerization sites along the entire subunit within all the structural domains were identified by a comprehensive mutational scan for co-trafficking with T1R3 in human cells. The data further reveal that the C terminus of the extracellular cysteine-rich domain needs to be properly folded for T1R3 dimerization and co-trafficking, but not for surface expression of T1R2 alone. These results guided the modeling of the T1R2-T1R3 dimer in living cells, which predicts a twisted arrangement of domains around the central axis, and a continuous folded structure between transmembrane domain loops and the cysteine-rich domains. These insights have implications for how conformational changes between domains are coupled within class C GPCRs.