Inverse Agonistic Action of 3-Iodothyronamine at the Human Trace Amine-Associated Receptor 5 (original) (raw)
Fig 2
Functional characterization of mouse and human TAAR5 in cAMP and IP3 assays.
(A) Cell surface expression was tested using an ELISA. Mouse and human TAAR5 were N-terminally HA-tagged and transiently transfected with COS-7 cells. Results are depicted as mean ± SEM obtained from 3 independent assays measured in 4 replicates. Data are presented as fold over mock transfection. An unpaired two-tailed t-test was performed which showed no significant difference of cell surface expression between both receptor orthologs. (B) HEK293 cells expressing mouse or human TAAR5 were stimulated with 100 μM DMEA. The cAMP accumulation was measured by competitive cAMP assay based on AlphaScreen technology. Results are depicted as either fold over basal mock or fold over DMEA stimulated mock transfection. Data are shown as mean ± SEM from n ≥ 3 independent experiments with 3 or more replicates. Statistical analyses were carried out with an unpaired two-tailed Welch-corrected t-test; ***p ≤ 0.001, compared to the respective basal activity. (C) HEK293 cells transiently expressing hTAAR5 or mTaar5 were incubated with or without 500 nM pertussis toxin (PTX), 20 hours prior to measurement. Supplement-free MEM was added to the untreated cell. IP3-luc levels were determined without (basal) and with PTX treatment. Results are presented as relative light units (RLU). Data were obtained from 3 to 6 independent experiments measured in triplicates and are shown as mean ± SEM. An unpaired two-tailed Welsh-corrected t-test was used for statistical analyses; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Human TAAR5 shows elevated basal IP3-luc activity, which was maintained after treatment with PTX pointing to a basal Gq/11 activity. Basal IP3-luc activity of mTaar5 was not significantly influenced by PTX treatment.