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).
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