PROJECT C4
Kinetics of metabotropic glutamate receptors
G-protein-coupled receptors (GPCRs) constitute the largest and pharmacologically most important family of cell surface receptors. They become activated by binding of agonists to the orthosteric binding site, but may also be activated or modulated via presumably allosteric binding sites. Upon activation, GPCRs trigger a signaling cascade that begins by coupling to their cognate G-proteins, followed by activation of these G-proteins by binding of GTP and subsequent activation of effector proteins via the G-proteins’ α– or βγ -subunits, respectively.
The kinetics of this signaling machinery have been the subject of investigation for many years. However, only for the very specific case of the light receptor, rhodopsin, the activation has been determined with certainty at ≈1 ms. For all other GPCRs it is still essentially unknown how fast they can be activated. Using different approaches and receptors, activation times ranging from tens of ms to minutes have been reported.
Our own measurements using fluorescently labeled receptors in intact cells and activation with agonists by superfusion, have given activation times of ≈50 ms. This value may well overestimate the true activation times of GPCRs, notably because it is derived from a large number of receptors on a cell (or part of a cell) which are not fully synchronized. We now intend to develop optimized systems to measure the true activation kinetics and to investigate whether non-visual GPCRs might approach the switch times of rhodopsin. To do so, we intend to develop three distinct but related strategies for
a) light-triggered activation via caged agonists,
b) light-triggered activation via tethered photolabile agonists, and
c) concentration-jump induced activation and deactivation by a piezo device.
In each case, the readout will be via fluorescence resonance energy transfer (FRET) for receptor activation, based on sensor constructs bearing various types of fluorophores. The data will be globally fitted by complex Markovian models to quantify the kinetics of binding and unbinding as well as activation and deactivation. On the long run, this strategy will be expanded from the initial receptor activation process to the entire signaling chain.
