GPR56,一种具有组成型和配体促进活性的粘附G蛋白偶联受体(aGPCRs),参与许多生理和病理过程。受体的组成型或配体促进的激活是否通过相同的分子机制发生,不同的激活模式是否导致G蛋白之间的功能选择性是未知的。在这里,我们显示GPR56组成型激活G12和G13。与3-α-乙酰氧基二氢脱氧骨杜宁(3αDOG)的组成型活化和活化不同,用抗体刺激,10C7,针对GPR56的胞外结构域(ECD)导致激活,使G13优于G12。一种自身蛋白水解缺陷型突变体,GPR56-T383A,也被10C7激活,表明通过自催化裂解暴露的束缚激动剂(TA),此激活模式不需要。相比之下,这种蛋白水解抗性突变体不能被3αDOG激活,表明两种配体的激活模式不同。我们显示N末端截短的GPR56构建体(GPR56-Δ1-385)缺乏组成活性,但被3αDOG激活。类似于3αDOG,10C7促进β-抑制蛋白2的募集,但GPR56内化是β-抑制蛋白独立的。尽管10C7的缓慢激活模式有利于G13而不是G12,但它有效地激活了BT-20乳腺癌细胞中的下游Rho途径。这些数据显示,不同的GPR56配体具有不同的激活模式,产生差异G蛋白选择性,但在异源表达系统和内源性表达受体的癌细胞中集中于Rho途径的激活。因此,10C7是研究GPR56活性的基础过程及其在癌细胞中的作用的有趣工具。
GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor\'s constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56\'s extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of β-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.