Agonist binding: a multistep process.

The structural basis of G protein-coupled receptor (GPCR) activation by agonists has been the focus of much experimental research and has inspired the generation of numerous kinetic and molecular models. In this issue, Liapakis et al. (2004) provide new mechanistic insight with an elegantly simple set of studies on a well characterized experimental system, the 2 adrenergic receptor ( 2AR). They provide evidence for the existence of one or more intermediate conformational states linking the inactive receptor to the fully active receptor. The authors determine the binding affinities and efficacies of a panel of ligands that represents the structural constituents of the catecholamine epinephrine (a subset is shown in Fig. 1A). By comparing the affinities of the different compounds, it is possible to determine the contribution of each chemical substituent of the ligand (catechol hydroxyls, -hydroxyl, N-methyl) to the binding affinity. The results suggest that there is no preformed binding site for the agonist epinephrine in the unliganded 2AR. Figure 1B illustrates what is meant by a preformed binding site. In this simple lockand-key model, all of the amino acid side chains that contribute to the binding site for epinephrine are in the optimal position to engage the chemical substituents of the ligand. The contribution of each substituent to the binding affinity will depend on the type of interaction between the substituent and the receptor (aromatic, van der Waal’s, hydrogen bonding, ion pairs). The interaction of each substituent with the binding site should make a significant contribution to ligand affinity, and this contribution should be relatively independent of contributions made by the other substituents. Based on these assumptions, binding affinity should increase by adding the catechol hydroxyls to phenethylamine to make dopamine, by adding the N-methyl to phenethylamine to make methylphenethlamine, or by adding the -hydroxyl to phenethylamine to make hydroxyphenethylamine. Yet the authors found similar binding affinities for all four compounds (phenethylamine, dopamine, methylphenethlamine, and hydroxyphenethylamine). Adding any one of the substituents (catechol hydroxyls, -hydroxyl, or N-methyl) to phenethylamine did not increase the binding affinity. However, the authors observed that the catechol hydroxyls, the -hydroxyl, or the N-methyl group increased the affinity by 10-fold when added to a ligand having any one of the other three substituents (considering the two catechol hydroxyls as one substituent). For example, adding the N-methyl group to dopamine makes methyldopamine. The affinity of methyldopamine for the 2AR is approximately 10 times greater than the affinity of dopamine. Moreover, each of these substituents contributed 60to 120-fold to binding affinity in ligands having both of the other two substituents. These binding studies do not support the simple model shown in Fig. 1B. One possible way to explain these results is a model whereby the agonist binds through a series of conformational intermediates as shown in Fig. 1C. In the unliganded state, there is a minimal, low-affinity binding site that permits interactions between the receptor and a few structural features on the agonist (e.g., the aromatic ring and the amine). Binding to this site increases the probability of a conformational transition that is stabilized by an interaction between the receptor and the catechol hydroxyls. The binding energy gained by interactions between the receptor and the catechol hydroxyls pays for the energetic costs of the conformational change. This conformational transition increases the probability of yet another conformational change stabilized by interactions between receptor and the -hydroxyl and/or the N-methyl. Thus, the receptor becomes activated through a series of conformational intermediates, and the energetic costs of receptor activation are paid in installments. This model is consistent with the results of studies using fluorescence spectroscopy to monitor agonist-induced conformational changes in the 2AR (Ghanouni et al., 2001; Swaminath et al., 2004). Fluorescence lifetime studies provide evidence for at least one intermediate conformational state in agonist bound receptor (Ghanouni et al., 2001). More recent time-resolved studies show that epinephrine induces a biphasic conformational change in purified 2AR, consistent with an intermediate conformational state (Swaminath et al.,