A hydrophobic cluster in the center of the third extracellular loop is important for TSH receptor signaling

Objectives: Previous reports on the follicle stimulating hormone receptor (FSHR) and choriogonadotrophic/luteinizing hormone receptor (CG/LHR), which belong to the glycoproteinhormone receptor (GPHR) family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of GPHRs, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the thyroid stimulating hormone receptor (TSHR).

Results: Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the specific role of five hydrophobic amino acids in the central portion of ECL3, and K660 at the ECL3/ transmembrane helix (TMH) 7 junction for TSHR signaling. Loss of Gs/Gq stimulation by mutants of K660 demonstrates the pivotal role of this position on TSHR conformation and signal transduction. By molecular modeling we determined potential interaction partners of K660: E409 and D410 in the N-terminus of TMH1 and D573 in the ECL2. Characterization of corresponding double mutants did not reveal hypothesized reconstitution of Gs/Gq-mediated signaling.

Conclusions: Therefore, K660 is not directly involved in a structural unit between ECL3 and the N-terminus of TMH1 or ECL2. The TSHR model suggests that the side chain of K660 is rather orientated toward the backbone of ECL2. Moreover, our findings provide evidence for a hydrophobic cluster, comprising residues 652–656 of ECL3, which seems to have a strong influence on intramolecular signal transduction and G-protein activation of the TSHR.