Lingerfelt Mary A, Zhao Pingwei, Sharir Haleli P, Hurst Dow P, Reggio Patricia H, Abood Mary E
Department of Chemistry and Biochemistry, University of North Carolina-Greensboro , Greensboro, North Carolina 27402, United States.
Center for Substance Abuse Research, Lewis Katz School of Medicine at Temple University , Philadelphia, Pennsylvania 19140, United States.
Biochemistry. 2017 Jan 24;56(3):473-486. doi: 10.1021/acs.biochem.6b01013. Epub 2017 Jan 11.
GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.
GPR55是一种新确定的A类G蛋白偶联受体，与炎症性疼痛、神经性疼痛、代谢紊乱、骨骼发育和癌症有关。迄今为止，已鉴定出的强效GPR55配体很少。这主要是由于缺乏关于GPR55显著特征的信息，例如对信号传导重要的残基以及参与GPR55信号级联反应的残基。这项工作的目标是确定对于GPR55内源性配体l-α-溶血磷脂酰肌醇（LPI）的信号传导以及GPR55激动剂ML184 {CID 2440433，3-[4-(2,3-二甲基苯基)哌嗪-1-羰基]-N,N-二甲基-4-吡咯烷-1-基苯磺酰胺} 的信号传导至关重要的残基。血清反应元件（SRE）和血清反应因子（SRF）荧光素酶测定被用作研究GPR55突变体处LPI和ML184信号传导的读数。基于最近的δ-阿片受体（DOR）晶体结构的GPR55 R*模型被用于解释所得的突变数据。发现两个残基对于GPR55处的激动剂信号传导至关重要，即K2.60和E3.29，这表明这些残基构成了ML184和LPI在GPR55处的主要相互作用位点。Y3.32F、H(170)F和F6.55A/L突变结果表明这些残基是ML184的正构结合位点的一部分，而Y3.32F和H(170)F突变结果表明这两个残基是LPI结合口袋的一部分。Y3.32L、M3.36A和F6.48A突变结果表明Y3.32/M3.36/F6.48簇在GPR55信号级联反应中的重要性。C(10)A和C(260)A突变表明这些残基在GPR55的细胞外结构域中形成了第二个二硫键，阻碍了配体在GPR55的跨膜螺旋1-跨膜螺旋7区域的细胞外进入。综上所述，这些结果提供了关于对LPI和ML184信号传导以及GPR55激活重要的GPR55残基的第一组离散信息。这些信息应有助于合理设计下一代GPR55配体，并有助于创建首个高亲和力GPR55放射性配体，这是该领域迫切需要的工具。
