Differenzierung molekularer Determinanten der G-Protein Selektivität des TSH-Rezeptors als Perspektive für neue Therapieansätze
Final Report Abstract
In this project we investigated molecular determinants of the thyrotropin receptor (TSHR) that are responsible for different activation mechanisms via G proteins and to determine their effect on the pharmacological profile of the receptor. The TSHR binds G proteins in a promiscuous manner and activates both Gs and Gq. In a first approach we studied the impact of the transmembrane helices (TMHs) for differences in Gs and Gq signaling by combining constitutively activating Gs mutations to double mutations with the goal to mimic additional conformational changes with subsequent effects for G protein signaling. We could show that most combinations led to an synergistic increased of constitutive Gs activity pointing towards a cooperative effect of the TMHs in the process of receptor activation. To our surprise, some double mutations revealed also ligand independent activation of the Gq pathway. For the first time we determined synergistic signaling effects for Gq and showed in contrast to previous observations for other receptors that combining single activating mutations are not restricted to a focused and limited subset of TMHs. Especially the Gq signaling profile of combination TMH1/2 is surprising, because these particular helices are connected via the intracellular loop (ICL) 1. So far, the ICL1 of G protein coupled receptors was not in focus for the activation of G proteins. As a consequence thereof, we investigated unknown details of (selective) interaction patterns at the intracellular receptor regions, with focus on ICL1. Utilizing the active opsin structure in complex we built a molecular model to study the TSHR/Gq heterotrimer complex. Several new amino acid contacts between the TSHR and G protein were suggested by this model, especially at ICL1 and helix 8. We performed model driven site-directed mutagenesis and characterized functional properties of the mutated receptors. The identified signaling sensitive amino acids were observed in our homology model to interact either directly with G proteins via H-bonds or indirectly affect G-protein activation via conformational changes within ICL1. Together with our recently published data of intracellular key-players for G protein coupling with the TSHR our results suggest a multiple contact interface between the TSHR and the G protein heterotrimer of Gq. All three ICLs of the TSHR contribute to the G-protein coupling process. Amino acids of ICL1 and the transitions between ICL1/TMH2, TMH3/ICL2 and ICL3/TMH6 as well as helix 8 potentially interact with Gαq, while the ICL1 also interacts with Gβγ. Interestingly our complex model of the TSHR - Gq heterotrimer was not only very similar to a GPCR complex model with Gq suggested by others few month after our publication. Moreover, very recently in May 2011 the crystal structure of an agonist bound GPCR complexed with the Gs heterotrimer was published and confirmed our suggested interaction of residues at ICL1 with the very end of the C-terminal helix α5. Taken together, GPCRs with promiscuous binding of G-protein subtypes (like the TSHR) are promising targets for investigating G-protein selectivity by studying determinants responsible for differentiated G protein activation.
Publications
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(2007). The TSH receptor is linked with AP1 and NFkappaB signaling in COS7 cells. Exp Clin Endocrinol Diabetes. 2007 Oct;115(9):590-3
Voigt C, Prodinger C, Paschke R
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(2008). Preferences of transmembrane helices for cooperative amplification of G(alpha)s and G (alpha)q signaling of the thyrotropin receptor. Cell Mol Life Sci. 2008 Dec, 65(24):4028-38
Jaeschke H, Kleinau G, Sontheimer J, Mueller S, Krause G, Paschke R
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(2010). Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor. PLoS One, 2010 Mar 18;5(3):e9745
Kleinau G, Jaeschke H, Worth CL, Mueller S, Gonzalez J, Paschke R, Krause G
