G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR–G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand–GPCR–partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gα_iβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gα_iβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

G 蛋白偶联受体 (GPCRs) 是治疗多种疾病的重要药物靶点。尽管 GPCR 的结构与结合的配体和 G 蛋白具有活性构象，但受体与其信号伙伴之间的详细分子相互作用仍然难以破译。为了解决这个问题，我们开发了一种高灵敏度、高通量基质辅助激光解吸/电离质谱 (MALDI-MS) 方法来询问信号转导的第一阶段。GPCR-G 蛋白复合物的形成被检测为配体对 GPCR 构象和偶联选择性的影响的代表。研究了超过 70 种配体-GPCR-伙伴蛋白组合，每个样品仅使用 1.25 pmol 蛋白质。我们确定了三种 GPCR（视紫红质、β-1 肾上腺素能受体 [β1AR] 和血管紧张素 II 1 型受体）对工程化 Gα 蛋白（mGs、mGo、mGi 和 mGq）和纳米抗体 80（Nb80 ）。我们发现在没有配体的情况下，GPCRs 可以结合 mGo，并且 G 蛋白 C 末端在 GPCR 识别中的作用是受体特异性的。我们使用 β1AR 举例说明了我们的量化方法，并证明了 Nb80 结合在帮助纳多洛尔向异丙肾上腺素置换中的变构作用。我们还用野生型异源三聚体 Gα 量化了复合物的形成 并且 G 蛋白 C 末端在 GPCR 识别中的作用是受体特异性的。我们使用 β1AR 举例说明了我们的量化方法，并证明了 Nb80 结合在帮助纳多洛尔向异丙肾上腺素置换中的变构作用。我们还用野生型异源三聚体 Gα 量化了复合物的形成 并且 G 蛋白 C 末端在 GPCR 识别中的作用是受体特异性的。我们使用 β1AR 举例说明了我们的量化方法，并证明了 Nb80 结合在帮助纳多洛尔向异丙肾上腺素置换中的变构作用。我们还用野生型异源三聚体 Gα 量化了复合物的形成_i βγ 和 β-arrestin-1 并表明卡维地洛诱导 β-arrestin-1 和 Gα _i βγ 与 β1AR 的耦合增加。标准化策略使我们能够定量测量 GPCR 与伴侣蛋白的结合亲和力。我们预计这种方法将广泛用于 GPCR 靶向药物的筛选和表征。