title: Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor.
abstract: The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str.10, 13125, Berlin, Germany. haas@fmp-berlin.de
authors.forename: Ann-Karin
authors.initials: AK
authors.lastname: Haas
authors.forename: Gunnar
authors.initials: G
authors.lastname: Kleinau
authors.forename: Inna
authors.initials: I
authors.lastname: Hoyer
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: Claudia
authors.initials: C
authors.lastname: Rutz
authors.forename: Ralf
authors.initials: R
authors.lastname: Schülein
authors.forename: Marvin C
authors.initials: MC
authors.lastname: Gershengorn
authors.forename: Gerd
authors.initials: G
authors.lastname: Krause
mesh_terms: D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D008024:Ligands; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP