Atypical Signaling and Functional Desensitization Response of MAS Receptor to Peptide Ligands

MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS.

MAS属于G蛋白偶联受体(G protein-coupled receptor, GPCR)，参与诸多生理过程的调控。已有研究表明，血管紧张素-(1–7)、血管紧张素片段及神经肽FF(neuropeptide FF, NPFF)等多种生理活性肽配体均可作用于MAS受体。目前针对肽配体刺激MAS受体后所引发的经典G蛋白信号转导及受体脱敏现象的相关研究仍较为有限。为此，本研究系统分析了肽配体激活MAS受体所介导的G蛋白信号通路。我们采用此前已被证实可激活G蛋白的MAS受体选择性非肽配体作为对照，在统一的细胞水平测定平台上开展对比实验。实验结果显示，非肽类激动剂激活MAS受体后可产生三类效应：一是升高细胞内钙水平与D-肌醇-1-磷酸(D-myo-inositol-1-phosphate, IP1)含量，该现象符合经典Gαq-磷脂酶C信号通路的激活特征；二是降低Gαi介导的环腺苷酸(cyclic adenosine monophosphate, cAMP)生成水平；三是激活Gα12依赖的荧光素酶报告基因表达。在所有上述检测体系中，MAS受体均表现出显著的组成型活性，且该活性可被非肽类反向激动剂所抑制。进一步的钙响应实验表明，当首次激活的效应消退后，MAS受体对第二次给予的非肽类激动剂刺激产生抵抗，提示其发生了功能性脱敏。与之形成鲜明对比的是，肽配体NPFF激活MAS受体后，仅会快速引发细胞内钙水平升高，而IP1的积累却极为微弱，这与经典Gαq-磷脂酶C信号通路的特征并不相符。同时，NPFF仅能微弱激活MAS受体介导的Gα12与Gαi信号通路。此外，与非肽类激动剂激活的MAS受体不同，经NPFF激活后的MAS受体可再次被激动剂有效刺激。针对关键配体结合位点的MAS受体突变体的功能实验结果表明，NPFF与非肽类配体的结合区域存在重叠。在非生理浓度(≥100 μM)下，血管紧张素-(1–7)及其他血管紧张素片段仅能微弱增强类似NPFF的钙响应效应。综上，本研究数据表明，肽配体可诱导MAS受体产生非典型信号转导与功能性脱敏现象。