title: Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.
abstract: The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, D-13125 Berlin, Germany.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Kleinau
authors.forename: Ann-Karin
authors.initials: AK
authors.lastname: Haas
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Worth
authors.forename: Inna
authors.initials: I
authors.lastname: Hoyer
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: Claudia
authors.initials: C
authors.lastname: Rutz
authors.forename: Marvin C
authors.initials: MC
authors.lastname: Gershengorn
authors.forename: Ralf
authors.initials: R
authors.lastname: Schülein
authors.forename: Gerd
authors.initials: G
authors.lastname: Krause
mesh_terms: D000495:Allosteric Site / Q000235:genetics*; D000596:Amino Acids / Q000378:metabolism*; D000242:Cyclic AMP / Q000032:analysis; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000596:Amino Acids; D008024:Ligands; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP