Standfirst
Using a directed evolution approach, researchers demonstrate a way of creating 'designer' receptors that are specifically activated by a ligand with no other biological activity in the cell.
G protein–coupled receptors (GPCRs) are an important class of membrane proteins that orchestrate a wide variety of cellular responses by binding environmental cues and relaying this information into the cell.
With an interest in investigating what individual neuron populations do in the brain, Bryan Roth and his colleagues at Case Western Reserve University School of Medicine and University of North Carolina Medical School set out to expand the toolbox for working with GPCRs. "The idea was that we could stick in an artificial receptor and turn those neurons off with a compound that does nothing [else in the cell], and that would tell us what the function of those neurons is in intact animals," says Roth.
His team set out to create a receptor, activated only by a pharmacologically inert compound, that they could express in any tissue or organ in the body to turn the activity of that tissue on or off. The ligand they chose for this purpose was clozapine-N-oxide (CNO). A related compound binds rat M3 receptors, so the researchers hypothesized that directed evolution of this receptor could be used to change its specificity. What they found experimentally, however, "is that you may want to start with an intermediate ligand which is equidistant between the native ligand and the ligand that you will eventually evolve the receptor toward, because you might not get it in one step; in our case, it did take repeated cycles of random evolution," says Roth.
Using mutagenic PCR, they produced libraries of modified M3 receptors and selected them in yeast for activation by an intermediate ligand. After several rounds of mutagenesis, the team obtained receptors that were activated by this ligand and then repeated the process to evolve the chosen mutants for CNO responsiveness.
As the goal was to use these receptors in mammalian cells, the researchers introduced the identified mutations into a human M3 receptor, and after screening this library of mutants in HEK T cells, they identified a double-mutant M3 receptor that was activated exclusively by CNO. As proof of principle, Roth and colleagues found that immortalized human pulmonary artery smooth muscle cells lacking the M3 receptor but expressing this designer receptor responded to CNO and effected downstream signaling. Also, in hippocampal neurons expressing a different designer receptor as well as an endogenous receptor, CNO specifically controlled the engineered receptor, inducing neuronal silencing—while the native receptor was unaffected.
Considering the wide range of natural ligands that bind various GPCRs, receptors could be engineered to be activated by essentially any ligand using this strategy. Other scientists are already using the receptors generated in this study as tools—with mice. But Roth sees other applications for these designer receptors: "Where we'd like to see it go further is not only using these as tools for addressing questions of interest to the basic scientist, but also to use these as therapeutic tools for tweaking the activity of engineered tissue in humans. And I think ultimately this is where this is going to go."