title: A hydrophobic cluster in the center of the third extracellular loop is important for thyrotropin receptor signaling.
abstract: Previous reports on the follicle-stimulating hormone receptor and choriogonadotropic/LH receptor, which belong to the glycoprotein hormone receptor family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of glycoprotein hormone receptors, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the TSH receptor (TSHR). Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the importance of five amino acids in the central portion of ECL3 and K660 at the ECL3/transmembrane helix (TMH) 7 junction for TSHR signaling. Decrease of G(s) activation and loss of G(q) activation by substitutions of K660 demonstrates a role for this position for TSHR conformation and signal transduction. By molecular modeling, we predicted potential interaction partners of K660:E409 and D410 in the N terminus of TMH1 and D573 in the ECL2. Complementary double mutants did not reconstitute G(s)/G(q)-mediated signaling, suggesting that K660 is not directly involved in a structural unit between ECL3 and the N terminus of TMH1. These results support a TSHR model in which the side chain of K660 is orientated toward the backbone of ECL2. Moreover, our findings provide evidence that a hydrophobic cluster, comprising residues 652-656 of ECL3, strongly influences intramolecular signal transduction and G protein activation of the TSHR.
authors.affiliation: Medical Department, University of Leipzig, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Claus
authors.forename: Holger
authors.initials: H
authors.lastname: Jaeschke
authors.forename: Gunnar
authors.initials: G
authors.lastname: Kleinau
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Gerd
authors.initials: G
authors.lastname: Krause
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D011989:Receptors, Thyrotropin; D008239:Lysine; D000409:Alanine