Role of cleavage and shedding in human thyrotropin receptor function and trafficking

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.