G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four G_s-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β₂ adrenergic receptors (β₂ARs) and β₁ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V₂ and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β₂AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

G 蛋白偶联受体 (GPCRs) 使用多种机制来调节丝裂原活化蛋白激酶 ERK1/2。β-Arrestins (βArr1/2) 是普遍存在的 G 蛋白信号传导抑制剂，可促进 GPCR 脱敏和内化，并作为 ERK1/2 激活的支架。使用 CRISPR/Cas9 删除 βArr1/2 和 G 蛋白的研究对 β-arrestins 在激活 ERK1/2 特定池中的作用提出了质疑。我们在三个不同的亲本和 CRISPR 衍生的 βArr1/2 敲除 HEK293 细胞对中比较了 siRNA 介导的 βArr1/2 敲低和用 βArr1/2 重组的影响，以评估 βArr1/2 缺失对 ERK1/2 激活的影响。 GS _{_}-耦合的 GPCR。在具有所有受体的所有亲本系中，对 βArr2 或 βArr1/2 特异的 siRNA 降低了 ERK1/2 刺激。相比之下，在不同细胞系之间和不同受体激活的情况下，使用 CRISPR 衍生的细胞系观察到不同的影响。对于 β ₂肾上腺素能受体 (β ₂ ARs) 和 β ₁ ARs，βArr1/2 缺失增加、减少或对不同 CRISPR 克隆中异丙肾上腺素刺激的 ERK1/2 激活没有影响。加压素 V ₂和促卵泡激素受体对 ERK1/2 的激活在这些细胞中减少，但通过用 βArr1/2 重组而增强。CRISPR βArr1/2 敲除细胞中脱敏和受体内化的丧失导致_β2AR 介导的 ERK1/2 刺激变得更加依赖于 G 蛋白，这通过重新引入 βArr1/2 被逆转。这些数据表明 βArr1/2 作为调节中心发挥作用，确定导致 ERK1/2 激活的机制不同途径之间的平衡，并注意不要将 βArr1/2 或 G 蛋白缺失细胞的结果外推至 GPCR 行为本机系统。