Kinetics of GPCR activation

While converging on the involvement of especially TM6 in receptor activation, the spectroscopic studies on rhodopsin and the p2 adrenergic receptor also indicated some possible important differences between the two receptors. The EPR spectroscopic read-outs for rhodopsin demonstrated a rapid formation of the active metarhodopsin II state (within microseconds) following light-induced conversion of the prebound cis-retinal to all-trans-retinal, whereas the conversion of metarhodopsin back to the inactive metarhodopsin III state was slow with a t1/2 of about 6 min (Farahbakhsh et al. 1993). In contrast to the rapid activation and the slow inactivation kinetics observed for rhodopsin, the spectroscopic analyses of the p2 adrenergic receptor indicated slow agonist-induced conformational changes (t1/2 ~ 2-3 min), significantly slower than the predicted association rate of the agonist (Gether et al. 1995, 1997b; Ghanouni et al. 2001b; Jensen et al. 2001). However, the reversal of the agonist-induced conformational change was relatively fast (t1/2 ~ 30 sec) (Gether etal. 1995,1997b; Ghanouni etal. 2001b; Jensen et al. 2001). These differences in activation kinetics could either reflect inherent differences between the two receptors or they could be a consequence of differences in how the changes were detected. Since the measurements were performed under similar conditions it is nevertheless more likely that they reflect differences between rhodopsin and a receptor activated by a 'diffusible' ligand. Of notable interest, several spectroscopic read-out have now been established either upon IANBD labelling of endogenous cysteines or cysteines at the cytoplasmic end of TM6 or upon labelling with fluoresceine maleimide in G protein coupling domain (Gether et al. 1995,1997b; Ghanouni etal. 2001b; Jensen etal. 2001). This consistency for the different read-outs, and the clear correlation between change in fluorescence and biological efficacy (Gether etal. 1995; Ghanouni et al. 2001b; Jensen et al. 2001) support the contention that the slow kinetics is an intrinsic property of the receptor, at least when conformational changes are being probed on purified protein using the sulfhydryl-reactive fluorophore, IANBD.

It is important to emphasize that our experiments have been carried out in the absence of G protein and that it is very likely that the G protein can affect the kinetics of the transition between the inactive agonist bound receptor complex (the AR state) and the active agonist bound receptor complex (the AR* state). Clearly, the slow kinetics of the agonist-induced conformational change in the absence of G protein would predict a high activation energy barrier for this transition (Gether et al. 1997a). It is conceivable that the G protein is able to stabilize the agonist-receptor complex and accordingly lower the activation energy barrier substantially and cause receptor activation to occur significantly faster. This may provide an explanation for the apparent discrepancy between the slow kinetics of agonist-induced conformational changes observed for the purified p2 adrenergic receptor with the rapid responses to agonist-stimulation of GPCRs in cells, such as, for example, activation of ion channels. Unfortunately, it has not yet been possible to test the kinetic importance of the G protein due to technical difficulties mostly due to instability of the GaS protein.

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