Wysolmerski Brian, Blythe Emily E, von Zastrow Mark
Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, San Francisco CA, USA.
Tetrad Graduate Program, University of California, San Francisco, San Francisco CA, USA.
bioRxiv. 2025 May 13:2025.05.12.653522. doi: 10.1101/2025.05.12.653522.
Many GPCRs trigger a second phase of G protein-coupled signaling from endosomes after initiating signaling from the plasma membrane. This inherently requires receptors to increase the concentration of active-state G proteins on the endosome membrane, but how this is achieved remains incompletely understood. We addressed this question by dissecting the regulation of G protein abundance and activity on endosomes following activation of several G-coupled GPCRs-the β2-adrenergic receptor, the VIP-1 receptor, and the adenosine 2b receptor-that are natively co-expressed and differ in their ability to internalize after activation. We first verify GPCR-triggered redistribution of Gα from the plasma membrane to a mixed population of intracellular membranes, including endosomes, that is both reversible after receptor inactivation and triggered irrespective of the ability of the GPCR to internalize. We next show that GPCRs trigger this redistribution process at native expression levels and describe a method, using conformational biosensors, to detect endosomal activation of endogenous Gα. Applying this method, we show that GPCR-mediated production of active-state Gα on endosomes depends on receptor endocytosis, whereas increasing the net amount of Gα on endosomes does not. Our results support a model for G regulation on endosomes mediated by two spatially separated receptor coupling events-one at the plasma membrane controlling endosomal G abundance and another at endosomes controlling G activity. Additionally, our results reveal location-bias in the selectivity of G protein activation on endosomes that is differentially programmed by GPCRs in a receptor-specific manner.
许多G蛋白偶联受体（GPCR）在质膜起始信号传导后，会在内体引发G蛋白偶联信号传导的第二阶段。这本质上要求受体增加内体膜上活性态G蛋白的浓度，但这一过程是如何实现的仍未完全清楚。我们通过剖析几种G偶联GPCR（β2肾上腺素能受体、VIP-1受体和腺苷2b受体）激活后内体上G蛋白丰度和活性的调节来解决这个问题，这些受体天然共表达，且激活后内化能力不同。我们首先验证了GPCR触发的Gα从质膜重新分布到包括内体在内的细胞内膜混合群体中，这种重新分布在受体失活后是可逆的，并且与GPCR的内化能力无关。接下来，我们表明GPCR在天然表达水平触发这种重新分布过程，并描述了一种使用构象生物传感器检测内源性Gα内体激活的方法。应用该方法，我们表明GPCR介导的内体上活性态Gα的产生依赖于受体内吞作用，而增加内体上Gα的总量则不然。我们的结果支持一种由两个空间分离的受体偶联事件介导的内体上G调节模型——一个在质膜控制内体G的丰度，另一个在内体控制G的活性。此外，我们的结果揭示了内体上G蛋白激活选择性的位置偏向性，这种偏向性由GPCR以受体特异性方式进行差异编程。
