Promiscuous G protein–coupled receptors (GPCRs) engage multiple Gα subtypes with different efficacies to propagate signals in cells. A mechanistic understanding of Gα selectivity by GPCRs is critical for therapeutic design, since signaling can be restrained by ligand–receptor complexes to preferentially engage specific G proteins. However, details of GPCR selectivity are unresolved. Here, we investigated cognate G protein selectivity using the prototypical promiscuous Gαq/11 and Gα12/13 coupling receptors, angiotensin II type I receptor (AT1R) and prostaglandin F2α receptor (FP), bioluminescence resonance energy transfer–based G protein and pathway-selective sensors, and G protein knockout cells. We determined that competition between G proteins for receptor binding occurred in a receptor- and G protein–specific manner for AT1R and FP but not for other receptors tested. In addition, we show that while Gα12/13 competes with Gαq/11 for AT1R coupling, the opposite occurs for FP, and Gαq-mediated signaling regulated G protein coupling only at AT1R. In cells, the functional modulation of biased ligands at FP and AT1R was contingent upon cognate Gα availability. The efficacy of AT1R-biased ligands, which poorly signal through Gαq/11, increased in the absence of Gα12/13. Finally, we show that a positive allosteric modulator of Gαq/11 signaling that also allosterically decreases FP–Gα12/13 coupling, lost its negative modulation in the absence of Gαq/11 coupling to FP. Together, our findings suggest that despite preferential binding of similar subsets of G proteins, GPCRs follow distinct selectivity rules, which may contribute to the regulation of ligand-mediated G protein bias of AT1R and FP.

混杂的 G 蛋白偶联受体 (GPCR) 以不同的功效与多种 Gα 亚型结合，在细胞中传播信号。GPCRs 对 Gα 选择性的机械理解对于治疗设计至关重要，因为配体-受体复合物可以抑制信号传导以优先接合特定的 G 蛋白。然而，GPCR 选择性的细节尚未解决。在这里，我们使用原型混杂 Gαq/11 和 Gα12/13 偶联受体、血管紧张素 II I 型受体 (AT1R) 和前列腺素 F2α 受体 (FP)、基于生物发光共振能量转移的 G 蛋白和通路选择性研究了同源 G 蛋白选择性传感器和 G 蛋白敲除细胞。我们确定，对于 AT1R 和 FP，G 蛋白之间的受体结合竞争以受体和 G 蛋白特异性方式发生，但对于其他测试的受体则不然。此外，我们发现 Gα12/13 与 Gαq/11 竞争 AT1R 偶联，而 FP 则相反，Gαq 介导的信号传导仅在 AT1R 处调节 G 蛋白偶联。在细胞中，偏向配体在 FP 和 AT1R 的功能调节取决于同源 Gα 的可用性。在没有 Gα12/13 的情况下，AT1R 偏向配体的功效增加，通过 Gαq/11 的信号很差。最后，我们表明 Gαq/11 信号的正变构调节剂也变构降低 FP-Gα12/13 耦合，在没有 Gαq/11 与 FP 耦合的情况下失去其负调制。一起，