The majority of constitutively activating hTSH receptor mutations are located in the transmembrane helices (TMH) as well as in the extra- and intracellular loops (ECLs, ICLs). S₂₈₁ is one of two positions in the ectodomain in which activating hTSHR mutations have been identified in vivo (S₂₈₁T, I, 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, H) showed expression similar to wild-type hTSHR and are tolerated at this position since they maintain basal activity or express only show slight constitutive activity. 3D-modeling of the hTSHR suggested S₂₈₁ 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 S₂₈₁ and ECL1, aromatic residues Y₂₇₉, Y₄₇₆, H₄₇₈, Y₄₈₁, Y₄₈₂ and H₄₈₄ were replaced by alanine. Functional characterization showed impaired cell surface expression and signaling for Y₂₇₉A and Y₄₈₁A in contrast to the other alanine mutants. However, substitutions of Y₂₇₉ and Y₄₈₁ with other aromatic residues exhibited surface expression and signaling comparable to wt hTSHR. Our results suggest that Y₂₇₉ in the ECD and likely Y₄₈₁ in the ECL1 also are involved in an aromatic environment around S₂₈₁ in the hTSHR which is important for a functional receptor conformation and intramolecular receptor signaling.