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首页> 外文期刊>Canadian Journal of Physiology and Pharmacology >Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ finger.
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Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ finger.

机译：与通过PDZ指连接的内皮素受体异二聚体相关的持续Ca2 +信号传导和延迟的内在化。

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G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.
机译：包括内皮素受体A（ETA）和B（ETB）在内的G蛋白偶联受体（GPCR）可能形成二聚体或高阶寡聚体，对信号传导产生深远影响。在这里，我们研究了在ETA C端内的PDZ手指基序及其在HEK293细胞中表达时在与ETB异源二聚化以及与自身同源二聚化中的作用。受体二聚化通过（i）蓝绿色荧光蛋白（CFP）（FRET供体）与融合到ET受体C-末端的四半胱氨酸/ FlAsH（FRET受体）之间的荧光共振能量转移（FRET）和（ii）共免疫沉淀来监测温和洗涤剂溶解后的ET受体。在苏氨酸403 /丝氨酸404处PDZ手指基序中的突变消除了FRET，并减少了异二聚体和同二聚体的共免疫沉淀。通过测量细胞内Ca2 +的动员和响应10 nmol / L ET-1攻击的受体内在化来评估功能后果。 PDZ突变将ETA：ETB异二聚体介导的持续Ca2 +信号转化为瞬时反应，类似于对同二聚体或单体的观察。可以看到，包含PDZ突变的异二聚体在与瞬时Ca2 +升高相似的时域（约5分钟）内化，并且与ETA同型二聚体或单体的内化具有相似的动力学。没有PDZ突变，异二聚体不会在超过15分钟的时间内内在化，这表明持续的Ca2 +信号传导是（至少部分地）延迟内在化的结果的一种有趣的可能性。结果与ETA-受体二聚化的结构模型一致，该结构模型将PDZ手指基序的苏氨酸403 /丝氨酸404置于异二聚体和同二聚体之间的相互作用界面上。持续的Ca2 +信号传导和ETA：ETB异二聚体的延迟内吞作用强烈主张影响跨膜信号传导和内在化的独特二聚体界面。

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