Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells

G‐protein‐coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid‐stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell‐surface TSHRs was apparently transient, but the reasons for the transient signaling by cell‐surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)‐based methods to precisely compare the kinetics of TSH binding and dissociation from cell‐surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible ( t 1/2,off =2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the β 2 ‐adrenergic receptor expressed in control cells. Enhancing TSHR internalization by β‐arrestin 2 overexpression did not modify the reversibility of TSHR‐cAMP signaling. These findings strengthen the view that the cointernalization of TSH‐TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.—Werthmann, R. C., Volpe, S., Lohse, M. J., Calebiro, D. Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells. FASEB J. 26, 2043‐2048 (2012). www.fasebj.org