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An Aromatic Environment in the Vicinity of Serine 281 Is a Structural Requirement for Thyrotropin Receptor Function

Third Medical Department, University of Leipzig (H.J., S.M., M.C., R.P.), 04103 Leipzig, Germany; National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (S.N.), Bethesda, Maryland 20892; and Leibniz-Institute for Molecular Pharmacology (G.Kl., G.Kr.), 13125 Berlin, Germany

Address all correspondence and requests for reprints to: Dr. Ralf Paschke, Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: ralf.paschke{at}medizin.uni-leipzig.de.

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. S₂₈₁ 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 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 wild-type hTSHR. Our results suggest that Y₂₇₉ in the extracellular domain and probably Y₄₈₁ in the ECL1 also are involved in an aromatic environment around S₂₈₁ in the hTSHR, which is important for functional receptor conformation and intramolecular receptor signaling.

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