Study Of The Structure And Induced Mutation Of Angiotensin Ⅱ And Endothelin-1 Receptor In Patients With Obliterative Vasculopathy

Objective: Obliterative vasculopathy represents pathological changes occuring in different diseases such as thromboangiitis obliterans(TAO) or Buerger’s disease1, preeclampsia, transplant rejection and systemic sclerosis(SSc). Typical of GPCRs, activation of Angiotensin II and Endothelin-1 receptors initiate several processes.we have developed GPCR activation assay, based on a yeast model already successfully used on the A2 B receptor, and expressed isolated AT1 R or ETAR. To investigate the effects of immune stimulation on the structure of the receptors, the preliminary step was to generate second extracellular loop(ECL2) mutations of the receptors and observe their influence on the receptor functioning stimulated with patient IgG or the endogenous ligands.Methods: In order to study the effects of immune stimulation on the ECL2 of both receptors, a GPCR activation assay was generated, using a yeast model which has already been successfully used on the A2 B receptor.In order to see whether the antibodies found in patients are binding to the second extracellular loops of the receptors and if this binding was specific to the amino acid sequence, we decided to swap the ECL2 of AT1 R and ETAR. Site-directed mutagenesis was performed to integrate the ECL2 of ETAR in AT1 R and the ECL2 of AT1 R in ETAR. Different MMY strains were tested to find the best to express AT1 R and ETAR and study their response to their endogenous ligands, Ang II and ET-1 respectively.Results: Growth was monitored and compared to the one of yeasts transformed with the wild-type receptor. In contrast to the absence of stimulation, Ang II and patient IgG activate significantly and dose-dependently wild type AT1 R dose-dependently; whereas AT1 R with the ECL2 of ETAR responded significantly less to both stimulations. Concerning ETAR, in contrast with the non-stimulated yeasts, ET-1 and patient IgG activated both wild type and ETAR with the ECL2 of AT1 R significantly and dose-dependently.Conclusion: The results suggest that AT1 R and ETAR might form constitutive heterodimers. Besides, IgG digestion brought convincing evidence that antibodies against AT1 R and ETAR play an important role in transducing intracellular signals. The results also confirmed that the second extracellular loop is a conformation-sensitive site for natural ligand and autoantibodies in AT1 R but not in ETAR. This result is thus a great advantage to understand the pathogenesis of immune-mediated obliterative vasculopathy and discover new therapeutic strategies.