Partial Agonism Detected on the Level of the Receptor

Many clinically relevant drugs actually act not as full agonists but rather as partial agonists on GPCRs. However, functional assays to determine the degree of agonism are very limited. In the past, only downstream cellular assays existed to study receptor activity, and such assays are dependent on the cellular context, as well as the expression level of the receptor. A significant problem with respect to GPCR expression levels is the existence of spare receptors (receptor reserve); if neglected, a spare receptor phenomenon may produce a seemingly full functional response for ligands that in fact are weak partial agonists. FRET offers the ability to study the degree of GPCR activity directly at the level of the receptor, and the approach may ultimately provide a means to more accurately determine the efficacy of ligands (see clonidine-induced FRET response in Fig. 10.4). In FRET assays, distance changes and/ or rotation of fluorophores due to ligand-induced conformational changes of the receptor is measured. How strong is the correlation between agonist-induced FRET change and change in activity of the receptor? If temporally resolved FRET-based receptor activation assays will eventually be robust enough to be introduced to high-throughput screening, a tremendous gain of functional information as well as kinetic information is possible.

How do we know that the change in FRET directly correlates with receptor activation? The agonist-induced FRET change between fluorophores attached to certain sites on the receptor reports changes in distance between these fluo-rophores and/or their orientation toward each other. That agonists induce these changes in FRET, whereas antagonists do not indicate that activation of receptors is somehow related to the observed change in FRET. However, it is possible that events subsequent to receptor activation, such as interactions with G proteins or arrestins or other unknown intracellular interaction partners, lead to bending of the fluorophores which then result in alterations of the FRET signal. A straightforward approach to strip associated proteins off the membranes is treatment with a 6-M urea containing buffer. For a2A-adrenergic receptor, the agonist-induced reduction in FRET was not altered in urea-stripped membranes [73]. Furthermore, the concentration dependence for agonist-mduced FRET changes was sensitive to the presence of nucleo-tide-free G proteins, and an excess of nucleotide-free G proteins increased the sensitivity of the FRET response toward agonist [73], a phenomena also observed with ligand binding studies. Interestingly, a reasonable correlation between the degree of agonism of partial agonist and the maximal achieved FRET change was also observed [33] .

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