%0 Journal Article %1 Castro2005 %A Castro, M. %A Nikolaev, V. O. %A Palm, D. %A Lohse, M. J. %A Vilardaga, J. P. %D 2005 %J Proc Natl Acad Sci U S A %K 1/chemistry/*metabolism Binding Dyes Energy Fluorescence Fluorescent Hormone, Hormone/metabolism Humans Kinetics Parathyroid Protein Resonance Structure, Tertiary Transfer Type Receptor %N 44 %P 16084-9 %T Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16236727 %V 102 %X Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).

@article{Castro2005, abstract = {Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Castro, M. and Nikolaev, V. O. and Palm, D. and Lohse, M. J. and Vilardaga, J. P.}, biburl = {https://www.bibsonomy.org/bibtex/215e76ea772cbcab5cf49d35b1c7d8dd9/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {0f2ab9e9fb211299526efeeadad9314b}, intrahash = {15e76ea772cbcab5cf49d35b1c7d8dd9}, issn = {0027-8424 (Print) 0027-8424 (Linking)}, journal = {Proc Natl Acad Sci U S A}, keywords = {1/chemistry/*metabolism Binding Dyes Energy Fluorescence Fluorescent Hormone, Hormone/metabolism Humans Kinetics Parathyroid Protein Resonance Structure, Tertiary Transfer Type Receptor}, month = {Nov 1}, note = {Castro, Marian Nikolaev, Viacheslav O Palm, Dieter Lohse, Martin J Vilardaga, Jean-Pierre Research Support, Non-U.S. Gov't United States Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):16084-9. Epub 2005 Oct 18.}, number = 44, pages = {16084-9}, shorttitle = {Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism}, timestamp = {2010-12-14T18:20:20.000+0100}, title = {Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16236727}, volume = 102, year = 2005 }