Zheng Chen, Weinstein Liana D, Nguyen Kevin K, Grewal Abhijeet, Gurevich Eugenia V, Gurevich Vsevolod V
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
bioRxiv. 2023 May 1:2023.05.01.538990. doi: 10.1101/2023.05.01.538990.
Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the role of arrestin-3 conformational equilibrium and of Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. Subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds, but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.
抑制蛋白可结合活性磷酸化的G蛋白偶联受体（GPCR）。在四种哺乳动物亚型中，只有抑制蛋白-3能促进细胞中JNK3的激活。在已有的结构中，抑制蛋白-3套索状环中的赖氨酸-295及其在抑制蛋白-2中的同源物赖氨酸-294直接与激活剂连接的磷酸基团相互作用。我们比较了抑制蛋白-3构象平衡以及赖氨酸-295在GPCR结合和JNK3激活中的作用。几个结合GPCR能力增强的突变体对JNK3的活性要低得多，而一个不结合GPCRs的突变体活性更高。突变体的亚细胞分布与GPCR募集或JNK3激活无关。赖氨酸-295的电荷中和及反向突变在不同背景下对受体结合有不同影响，但对JNK3激活几乎没有影响。因此，GPCR结合和抑制蛋白-3辅助的JNK3激活有不同的结构要求，这表明促进JNK3激活是未与GPCR结合的抑制蛋白-3的功能。
