Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin ReceptorReport as inadecuate

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In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors GPCR and different subtypes of G-proteins. The thyrotropin receptor TSHR binds G-proteins promiscuously and activates both Gs cAMP and Gq IP. Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop ICL 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits Gα as well as Gβγ. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 L440, T441, H443 and helix 8 R687 that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only.Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR-Gq-heterotrimer complex is characterized by more selective interactions than the TSHR-Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.

Author: Gunnar Kleinau, Holger Jaeschke, Catherine L. Worth, Sandra Mueller, Jorge Gonzalez, Ralf Paschke, Gerd Krause



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