G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization¹. Although it is unclear how GRKs recognize these receptors^2,3,4, a conserved region at the GRK N terminus is essential for this process^5,6,7,8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.

G 蛋白偶联受体 (GPCR) 激酶 (GRK) 选择性磷酸化激活的 GPCR，从而启动它们进行脱敏¹。虽然尚不清楚 GRK 如何识别这些受体^2,3,4，但 GRK N 末端的一个保守区域对该过程至关重要^5,6,7,8. 在这里，我们报告了一系列与视紫红质激酶 (GRK1) 结合的光激活视紫红质 (Rho*) 的冷冻电子显微镜单粒子重建，其中 GRK1 的 N 末端形成一个螺旋，停靠在 Rho* 的开放细胞质裂隙中. 该螺旋还针对 GRK1 激酶结构域进行包装，并将其稳定在活性结构中。大多数 GPCR 中保守的碱性残基与 GRK 中保守的酸性残基之间的静电相互作用进一步稳定了该复合物。我们没有观察到 GRK1 的 G 蛋白信号同源结构域或视紫红质 C 末端的调节剂的任何密度。与质谱分析的交联证实了这些结果，并揭示了受体结合的 GRK1 中的动态行为，这将允许受体尾部多个位点的磷酸化。我们已经确定了 GRK1 残基，其突变增强了激酶活性并与 Rho* 交联，以及参与酸性磷脂激活的残基。根据这些数据，我们提出了一个通用模型，说明一小部分蛋白激酶如何识别并被数百种不同的 GPCR 激活。