An aromatic environment in the vicinity of serine 281 is a structural requirement for thyrotropin receptor function

The majority of constitutively activating human TSH receptor ( hTSHR) mutations are located in the transmembrane helices as well as in the extracellular (ECLs) and intracellular loops. S-281 is one of two positions in the ectodomain in which activating hTSHR mutations have been identified in vivo (S281T, I, and N). To investigate the functional properties of this key residue in more detail, S281 was replaced by each of the other 19 amino acids. Many substitutions led to constitutive receptor activation, suggesting that S281 plays a pivotal role in maintaining the receptor in its inactive state. Strikingly, all substitutions with aromatic residues (S281W, F, Y, and H) show expression similar to that of wild-type hTSHR and are tolerated at this position because they maintain basal activity or express only slight constitutive activity. Three-dimensional modeling of the hTSHR suggested that S-281 and surrounding residues are in close proximity to ECL1. To investigate the possible importance of an aromatic environment between the ectodomain in the vicinity of S-281 and ECL1, aromatic residues Y-279, Y-476, H-478, Y-481, Y-482, and H-484 were replaced by alanine. Functional characterization showed impaired cell surface expression and signaling for Y(279)A and Y(481)A, in contrast to the other alanine mutants. However, substitutions of Y279 and Y481 with other aromatic residues exhibited surface expression and signaling comparable to wild-type hTSHR. Our results suggest that Y-279 in the extracellular domain and probably Y-481 in the ECL1 also are involved in an aromatic environment around S-281 in the hTSHR, which is important for functional receptor conformation and intramolecular receptor signaling.