To map the potential interacting residues we first performed in-silico studies of the TSHR-TMD using a homology model and employing Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4, TM2 made contact with TM5 and TM2 made contact with TM5.

In order to confirm the proximity of these contact residues we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies.

Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming "domains of attraction" in a cooperative manner that is not dominated by interactions between specific residues.