A comment on the interpretation of ligand binding experiments for agonists

Ligand binding experiments are widely used by pharmacologists, biochemists and structural biologists to garner information about the physical interactions between ligands and their receptors. However, it is often overlooked that these experiments do not have the same interpretation for agonists as they do for antagonists – in the case of agonists, they cannot provide the information which is so often assumed of them (Colquhoun, 1987, 1998). Here, I discuss some under-appreciated problems with the interpretation of equilibrium competition binding experiments for agonists.

Application of the law of mass action to Scheme 1 provides the derivation for Equation 1, thus homologous competition binding experiments work well for antagonists. The simplest mechanism which can describe the homologous competition between a labelled (L) and unlabelled (U) agonist is

As such, homologous competition binding experiments cannot provide agonist K_D, but rather provide a macroscopic equilibrium constant (macroscopic affinity; the agonist concentration required to occupy half of the binding sites) which is dependent on the microscopic equilibrium constants for affinity (K_D) and efficacy (E). This makes comparisons of the affinity of antagonists and agonists difficult (because a microscopic affinity is compared with a macroscopic affinity), and shows that a change in agonist binding cannot be attributed to a change in K_D based on a binding experiment alone (Colquhoun, 1998; Higham & Colquhoun, 2024).

The same is true for heterologous competition binding experiments. Consider the competition between a labelled antagonist (L) and an unlabelled agonist (U), thus

It is patently clear that the mechanisms in Schemes 1-3 are too simple to describe any real receptor. Consequently, the interpretation of real binding data will be even less straightforward than highlighted by the simple mechanisms discussed so far.

As in the simpler case in Scheme 3, the microscopic affinity (K_U) of the competing agonist (for the R state) cannot be isolated because binding is dependent on other factors; namely, the agonist's efficacy and the receptor's constitutive activity. Only the macroscopic affinity of the agonist (given here by $K_U\frac{1+E_0}{1+E_U}$) can be found using binding experiments – a property which, unlike the microscopic affinity, has no simple interpretation. Analysis of the two-state mechanism for two competing neutral antagonists $(E_L=E_U=E_0)$ shows that ${\left[U\right]}_{50}=K_U(1+c_L)$, reiterating the validity of the Cheng-Prusoff equation for finding antagonist affinity.

From the foregoing discussion, it is clear that agonist binding at GPCRs (and other types of receptor) depends on multiple factors and not just the initial binding step, making the interpretation of binding experiments difficult. Experimental evidence has shown that the binding of many GPCR agonists is biphasic, with a high- and low- affinity component, and is sensitive to the presence of guanosine triphosphate (GTP) (for review see Colquhoun, 1998; Strange, 2008). The high-affinity component of binding is absent when experiments are performed in the presence of GTP or a non-hydrolysable analogue. It has been proposed that this may be due to the high-affinity active state of the receptor, or a similar active-like state, being stabilised by the binding of G-protein, giving rise to two receptor populations; free receptors (largely R, low-affinity) and those bound by G-protein (largely R*, high-affinity). In the presence of GTP, rapid G-protein dissociation can take place upon receptor activation (R*). Consequently, there is likely to be a paucity, though not an absence, of the GR* and UGR* states in the presence of GTP (i.e., G has a very low affinity for R*; K_G* and K_UR* are very large). While this will reduce the overall coupling between receptor and G-protein, an appreciable fraction of the receptor population will still be made up of GR (depending on E_GR) and UGR (depending on E_UGR) at lower agonist concentrations. The fraction of receptors occupying the GR and UGR states will decrease as E_GR and E_UGR increase because a greater fraction of receptors will occupy the GR* and UGR* states from which G rapidly dissociates. At a saturating agonist concentration – assuming that K_G* and K_UR* are very large, and that E_GR > E₀ and E_UGR > E_U – binding in the CTC mechanism is dominated by UR* and, to a lesser extent, UGR (UR* becomes increasingly dominant as agonist efficacy increases).

On the basis of the discussion above, it is clear that care is required when interpreting and reporting the results of binding experiments for agonists because they very often cannot provide the information (i.e., microscopic equilibrium dissociation constants) which is assumed of them. This may be particularly problematic in binding experiments using intact cells, as opposed to membrane preparations, wherein the assay conditions are less easily controlled.

James P. Higham: Conceptualization (equal); formal analysis (equal); writing—original draft (equal); writing—review and editing (equal).

I declare that I have no competing interests, financial or otherwise.