Introduction to DREADDs – Control Over G Protein Coupled Receptor GPCR signaling

Gee, Protein, What Do You Do?

Manipulation of a system under investigation is the backbone of experimentation. A new tool called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows us to hijack cell signaling and study cell function within living organisms. Like its cousin technique, optogenetics, DREADD technology uses a viral vector to introduce a mutant G-protein coupled receptor (GPCR) encoding gene into cells of interest. You select your target cells targeted by using different promoters in the viral gene construct. After waiting two to three weeks for expression of the mutant receptor in infected cells, you activate themodified GPCR using clozapine-N-oxide (CNO), a synthesized drug. GPCR signaling directs a number of important cellular processes, so being able to manipulate to assess function is pivotal.

Advantages of DREADDs

DREADDs allow researchers to gain control over GPCR signaling and offer several advantages over optogenetic manipulations of cell activity. One advantage of using DREADDs is that you can flip the cellular switch on or off in vivo simply by injecting CNO systemically – no implantation of fiber optic arrays necessary. You only affect the local cell populations expressing your DREADD virus and you can use multiple virus injections to cover larger cell populations.1 Optogenetics is limited to where you direct the activating laser.  This makes manipulation of large cell populations difficult.

Compared to the sub-second time scale of optogenetic manipulation, one might view the slower time scale of DREADDs as a limitation. Yet, others argue that DREADDs manipulate cellular activity in a much more naturalistic way compared to optogenetics.1  This is because it uses the cells’ native GPCR signaling cascades, instead of rapidly opening artificial channels to let ions flow into the cell in response to light bursts. DREADDs’ control of slower GPCR signaling cascades allows you to study longer term cellular processes.2 CNO is also cleared reasonably rapidly.  This allows temporal control and the possibility of repeat dosing.1

DREADD technology holds a lot of promise for cell function studies, but its development was born from a long history of trying to engineer designer receptors.

The Intracellular Space Race

Several different technologies over the last 25 years attempted to gain control over GPCR signaling. The first attempt produced a mutant ?2-adrenergic receptor that did not respond to its native agonist hormones.3 This GPCR was activated instead by synthesized butanone derivative, L-185,870. However, the synthesized drug had low receptor affinity and its metabolic properties in a living creature were unknown, making it unsuitable for use in vivo.4

Using a similar approach, researchers engineered receptors activated solely by synthetic ligands (RASSLs) by mutating kappa-opioid receptors (Gi-coupled) to render them unresponsive to their native ligand, dynorphin.5 After that, many different RASSLs were engineered using modified serotonin, histamine, and melanocortin receptors.4 However, their ligands had activity at endogenous receptors and not just at the RASSLs. Other problems included \low receptor affinity of the ligand and high constitutive activity.

DREADDs Fit the Bill

Finally, DREADDs were developed by screening massive numbers of muscarinic receptor mutants for low constitutive activity, high ligand affinity, and specificity to the synthesized ligand.6 These mutant genes code for muscarinic receptors that are no longer activated by their native agonist, acetylcholine. Instead, activation of these modified GPCRs occurs in the presence of very low concentrations of CNO, an otherwise biologically inert drug. A family of DREADDs has been generated that couple to different types of G-proteins – Gi, Gs, and Gq. You can select the appropriate DREADD for activation or inactivation of intracellular signaling within the infected cells. DREADDs are now the most widely used G-Protein signaling hack. They are especially useful in neuroscience as CNO readily crosses the blood-brain-barrier to reach the central nervous system.

Validating Future DREADD Use

The challenge to using this technology, as is true with many, is that you must verify that your manipulation is doing what you intended. The NIH is requiring that new grants address issues of technique validation. This not only promotes better, more meticulous science, but also may help curb the current so-called “replication crisis”.

There is increasing awareness that validation is a part of using DREADDs successfully. You should determine which cell types your virus is in so that the functional outcome of GPCR signaling is interpretable. You also need to verify activation of G-protein signaling when you apply the designer drug. This is accomplished by electrophysiology in neurons or by quantifying downstream signaling intermediates (like cAMP, phospholipase C, and calcium) using more traditional techniques. Finally, you must also use control viruses that either 1) do not express the receptor gene or 2) that express receptors non-responsive to CNO.

As with any new technology, there are bound to be unforeseen challenges to be tackled. Yet no one can deny that DREADDs are quite useful to have in your tool belt.

Further Reading

  1. Smith KS, Bucci DJ, Luikart BW, Mahler SV. (2016) DREADDs: use and application in behavioral neuroscience. Behav. Neurosci. 130(2): 137–55.
  2. Lopez AJ, Kramar E, Matheos DP, White AO, Kwapis J, Vogel-Ciernia A, et al. (2016) Promoter-specific effects of DREADD modulation on hippocampal synaptic plasticity and memory formation. J. Neurosci. 36(12): 2588–99.
  3. Strader CD, Gaffney T, Sugg EE, Candelore MR, Keys R, Patchett AA, Dixon RA. (1991) Allele-specific activation of genetically engineered receptors. J. Biol. Chem. 266(1): 5–8.
  4. Conklin, B.R., Hsiao, E.C., Claeysen, S., Dumuis, A., Srinivasan, S., Forsayeth, J.R., et al. (2008) Engineering GPCR signaling pathways with RASSLs. Nature Methods 5(8): 673–8.
  5. Coward P, Wada HG, Falk MS, Chan SDH, Meng F, Akil H, et al. (1998) Controlling signaling with a specifically designed Gi-coupled receptor. Proc. Natl. Acad. Sci. 95: 352–7.
  6. Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL. (2007) Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. 104(12): 5163-5168.
Image Credit: Phylogeny Figures

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