Figure 6.
Mutation of arginine 3.36 eliminates agonist function of zaprinast, kynurenic acid and compound 10. (A, B) Receptor-β-arrestin-2 interaction BRET assays in response to varying concentrations of kynurenic acid (A) or zaprinast (B) were performed as in Figure 4 using either wild-type or Arg3.36Ala mutants of either FLAG-hGPR35-eYFP or FLAG-rGPR35-eYFP. The effects of the Arg3.36Ala mutations on response to a single concentration (10−4 M) of compound 10 that was maximally effective at the wild-type GPR35 orthologues was also assessed (C). Total levels of GPR35 expressed, as monitored by eYFP fluorescence above non-transfected cells, were unaltered by the Arg3.36Ala mutation (D), while cell surface delivery was monitored by anti-FLAG elisa (E) of the forms of GPR35. * Less than wild-type P < 0.05. (F) The amino acid sequence of a section of transmembrane domain III from both human and rat GPR35 is shown with Arg3.36 highlighted.