Fig. 5: The chimeric approach allows Nb6 to bind to 5-HT2AR and ETA.
From: Nanobody-enabled monitoring of kappa opioid receptor states
a Schematic of GPCR-KOR chimera generation. b BRET assays showing that 5-HT2AR-KOR or ETA-KOR chimera can bind to Nb6. Agonist 5-HT or Endothelin-1 causes dissociation of Nb6 from the chimeric receptor 5-HT2AR or ETA, respectively. EC50 values were summarized in Supplementary Table 4. (N = 3, three experiments each done in duplicate). c BRET assays showing that chimeric receptor 5-HT2AR-KOR or ETA-KOR can activate canonical G protein coupling similarly compared to the wild type receptors. EC50 values were summarized in Supplementary Table 5. (N = 3, three experiments each done in duplicate). d A comparison between Nb6 and miniGq proteins in reporting activation and inactivation in 5-HT2AR or ETA in the presence of agonists and antagonists. Experiments were conducted in the BRET assay. The first and second arrow indicates the time when the agonists and antagonists were added, respectively. For different receptors tested, the agonist and antagonist were 5-HT/Risperidone for 5-HT2AR, and Endothelin-3/CI 1020 for ETA. The reason that Endothelin-3 was used for ETA here instead of Endothelin-1 is that Endothelin-1’s affinity is too high to be replaced off. e BRET assays show that Nb6 can also bind to ‘orphan’ GPCRs in their basal state. ***p < 0.001 as compared to wild type group, unpaired t test. Error bars represent SEM. (N = 4, four experiments each done in duplicate). Source data are provided as a Source Data file.
