About 30% of current drugs target G protein-coupled receptors (GPCRs) [76
] or their associated protein network. ET was created specifically to study this pathway, which underlies smell, taste, vision, pain and much of endocrine and autonomic pharmacology. One goal is to identify and then rationally modify the molecular basis of signaling to identify novel possible therapeutic targets. Thus, following the same type of protein redesigns as above, separation of function mutations in the receptor [77
], Gα [59
], and Regulator of G protein Signaling (RGS) [74
] validated prior predictions of interaction sites for export, receptor coupling [58
], and downstream effector activation [65
], respectively. Functional rewiring was demonstrated in the RGS case by switching top-ranked cognate residues among homologs [74
]. And a peptide designed to mimic the top-ranked residues of a novel site in G protein receptor kinases impaired GPCR phosphorylation, confirming a role in this interaction [68
]. All these studies target top-ranked ET residues at, or near, the surface of the protein.
However, evolutionary analysis can also inform the internal mechanisms of a protein. A trace of visual rhodopsins versus the broader set of rhodopsin-like GPCRs identified two separate structural subdomains buried in the core of the seven-helical transmembrane bundle [78
]. The first one, unique to rhodopsins, was a putative ligand-specific binding site. The other one, common to all GPCRs, was a putative evolutionary conserved allosteric pathway that, over a distance, transforms ligand binding into effector activation. As predicted, point mutations to these sites then respectively impaired ligand binding or caused constitutive activity [78
]. Moreover, three mutations in the allosteric pathway near the G protein coupling site blocked G protein signaling but kept the β-arrestin signaling intact [79
]. These studies highlight the existence of functional modules within the structure and how they may be exploited to effectively sever just one of the two signaling branches efferent from activated receptors. More recently, related work investigated the correlations between sequence positions within a protein family and found similar structure-function partitioning of the protein into groups of residue positions referred to as “protein sectors” [80
]. This analysis was extended to the S1A serine proteases and found mutations of the individual “protein sectors” lead to different effects focused either on catalytic power or thermal stability.
To test further how well such evolutionary modules guide protein engineering, and to understand the origin of ligand-biased signaling, whereby different ligands signal via G proteins or β-arrestin to different extents, a study swapped top-ranked ET residues from the putative common allosteric pathway between two antagonistic psychoactive receptors, those for serotonin and dopamine [81
], . All single point mutants were expressed and bound normally to a bioamine antagonist. Strikingly, all of them also exhibited altered binding or signaling, by either dopamine or serotonin, and these effects were mostly separable or even paradoxical. Notably, four mutants significantly enhanced serotonin response without increasing serotonin binding. And two of these four mutants had decreased dopamine signaling, even though dopamine affinity was as good or better than in the wild type receptor.
Determinants of allosteric specificity in psychoactive receptors
This independent reprogramming of binding and of signaling from dopamine to serotonin highlights allosteric specificity, namely, the pathway itself can determine which bound ligands signal, separate of binding site affinity. Moreover, the key determinants of the allosteric pathway can be traced evolutionarily and then recoded, one top-ranked ET residues at a time–much as the tumblers of a lock are rekeyed. Presumably, during evolution, single mutations constantly change ligand affinity, effector biases, or the wiring between them, and thus probe alternative wiring at GPCR network nodes. In practice, many of the key residues surround structural waters, as shown in , suggesting a potential site where a drug could influence ligand-biased signaling [81