Funktion und Struktur von rezeptorinternem Transmitter (rIT) und multipler intramolekularer Aktivierungsmechanismus des TSH-Rezeptors
Final Report Abstract
Clarification of the intramolecular activation and signaling steps of the TSHR is the core issue for developing therapeutic approaches in the field of thyroid diseases. Therefore, this project was designed to obtain more information about the receptor activation mechanisms. Utilizing information around the TSHR we substantially expanded our information system to analyse sequence structure function relationships of glycoprotein hormone receptors (www.ssfa-gphr.de). The systems served in this project as a backbone for concept developments and mutant selections. The outcome of the present project has shed light on the particular function of structural determinants in the initial steps of receptor activation such as: 1) hormone binding at the extracellular site; 2) hormone interaction at a second binding site in the hinge region (HinR); 3) signal regulation via sequence motifs in the HinR; and 4) synergistic signal amplification by cooperative effects of the extracellular loops toward the transmembrane region. In detail, our study revealed that besides binding at the LRR, TSH also interacts with specific residues in the HinR. Therefore, we can conclude that the HinR is not an inert extracellular linker but rather appears to be an important structural and functional link between ligand binding and subsequent receptor activation. In detail, we could show that besides the known binding sites within the LRR and the sulfation site Y385 further 13 HinR positions are involved in ligand binding. These multiple interaction sites are scattered along the HinR whereas a cluster of binding sites seems to exist at region F381-D386. Among the identified positions, seven charged residues could be detected, which obviously participate in hormone binding via charge-charge interactions. Using the human TSH analog TR1401 we provide evidence for potential electrostatic interactions between the negatively charged HinR residues E297, E303 and D382 and positively charged residues of the superagonisitc bovine TSH and TR1401 in contrast to human TSH. These interactions seem to be the molecular cause for the higher potency and efficacy of the tested TSH variants in comparison with human TSH. Nevertheless, HinR-TSH interactions are further based on hydrophobic and hydrophilic residues at HinR that are necessary to maintain hormone-receptor binding. In addition to previous evolutionary strategies focusing only on the hormone, these results provide a complementary approach for a receptor-driven generation and optimization of orthosteric antagonists, inverse agonists, and also improved TSH analogs focusing on the binding and activation relevant HinR. Moreover, these new binding relevant HinR positions and the identified charge-charge interactions with the hormone provide new detailed information for the orientation of TSH toward the HinR. The results of our receptor-based investigation of TSH-TSHR interactions and especially the provided specific characteristics of the HinR binding sites should lead to further studies, which will disclose the direct interaction partners at the hormone molecule. Due to the sequence homology between TSHR, LHCGR and FSHR, such findings should also provide further insights into the molecular basis of GPH-GPHR interactions. Furthermore, we could show that amino acids between T257 and Y279 located in the transition between LRR and TSHR are important for TSHR folding, probably developing an additional leucinerich repeat. Based on our signaling results of mutations in this region, we suggest an extracellular activation mechanism that supports an intramolecular agonistic unit as a central switch for activating effects at the extracellular region toward the serpentine domain. Our observations analyzing simultaneous combination of constitutively activating mutations in the extracellular region of the TSHR support the concept that many amino acids in this region participate in full GPHR activation and signaling, which presumably result from multiple hormone contacts simultaneously at both the LRRD and the HinR. These results support a model in which their proximity allows residues at the positions of CAMs and inactivating mutations to function together to transmit the extracellular signal toward the TMHs to convert the TSHR to its active conformation. The results of this project provide new information regarding the structural features and functionalities of extracellular TSHR regions and therefore contribute to understanding the dynamics and mechanisms of TSHR activation, which is the cornerstone for the design of new therapeutic approaches in the field of thyroid diseases as targeted pharmacological interventions directly affecting the TSHR.
Publications
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(2008). Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor. FASEB J. 22(8):2798-808
Kleinau G, Jaeschke H, Mueller S, Raaka BM, Neumann S, Paschke R, Krause G
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(2008). Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation. J Biol Chem. 283(26):18048-55
Mueller S, Kleinau G, Jaeschke H, Paschke R, Krause G
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(2009). The superagonistic activity of bovine thyroid-stimulating hormone (TSH) and the human TR1401 TSH analog is determined by specific amino acids in the hinge region of the human TSH receptor. J Biol Chem. 284(24):16317-24
Mueller S, Kleinau G, Szkudlinski MW, Jaeschke H, Krause G, Paschke R
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(2009). Thyrotropin- and Homologous Glycoprotein Hormone Receptors: Structural and Functional Aspects of Extracellular Signaling Mechanisms. Endocr Rev. 2009 Apr; 30(2):133-51
Kleinau G, Krause G
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(2010). An Interactive Web-Tool for Molecular Analyses links Naturally Occurring Mutation Data with Three-Dimensional Structures of the rhodopsin-like Glycoprotein Hormone Receptor. Human Mutation, Jun; 31(6): E1519-25
Kleinau G, Kreuchwig A, Worth CL, Krause G
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(2010). The hinge region: an important receptor component for GPHR function. Trends Endocrinol Metab. 21(2):111-22
Mueller S, Jaeschke H, Günther R, Paschke R
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(2011). Defining structural and functional dimensions of the extracellular thyrotropin receptor region. J Biol Chem. 286(25):22622-31
Kleinau G, Mueller S, Jaeschke H, Grzesik P, Neumann S, Diehl A, Paschke R, Krause G
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(2011). Identification of novel TSH interaction sites by systematic binding analysis of the TSHR hinge region. Endocrinology. 152(8):3268-78
Mueller S, Szkudlinski MW, Schaarschmidt J, Günther R, Paschke R, Jaeschke H
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(2011). Research resource: Update and Extension of a glycoprotein hormone heceptors web application. Mol Endocrinol. 2011 Apr;25(4):707-12
Kreuchwig A, Kleinau G, Kreuchwig F, Worth CW, Krause G
