Optical Approaches for Investigating Neuromodulation and G Protein-Coupled Receptor Signaling

Abstract

Despite the fact that roughly 40% of all US Food and Drug Administration (FDA)-approved pharmacological therapeutics target G protein-coupled receptors (GPCRs), there remains a gap in our understanding of the physiologic and functional role of these receptors at the systems level. Although heterologous expression systems and in vitro assays have revealed a tremendous amount about GPCR signaling cascades, how these cascades interact across cell types, tissues, and organ systems remains obscure. Classic behavioral pharmacology experiments lack both the temporal and spatial resolution to resolve these long-standing issues. Over the past half century, there has been a concerted effort toward the development of optical tools for understanding GPCR signaling. From initial ligand uncaging approaches to more recent development of optogenetic techniques, these strategies have allowed researchers to probe longstanding questions in GPCR pharmacology both in vivo and in vitro. These tools have been employed across biologic systems and have allowed for interrogation of everything from specific intramolecular events to pharmacology at the systems level in a spatiotemporally specific manner. In this review, we present a historical perspective on the motivation behind and development of a variety of optical toolkits that have been generated to probe GPCR signaling. Here we highlight how these tools have been used in vivo to uncover the functional role of distinct populations of GPCRs and their signaling cascades at a systems level. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) remain one of the most targeted classes of proteins for pharmaceutical intervention, yet we still have a limited understanding of how their unique signaling cascades effect physiology and behavior at the systems level. In this review, we discuss a vast array of optical techniques that have been devised to probe GPCR signaling both in vitro and in vivo.

Fig. 1

Photocycles of GPCR opsins. Schematic demonstrating photocycles for (A) bleaching, (C) reverse, (D) bistable, and (D) chimeric opsins. Cis-retinol bound receptors are in the “off state” in complex with their cognate G proteins. For bleaching opsins (in this case, rhodopsin), (A), after exposure to light, the receptor-bound cis-retinol undergoes cis-trans isomerization, leading to a conformational change in the receptor to its “on state.” This change in turn catalyzes the exchange of GDP for GTP in the cognate heterotrimeric G protein, leading to its dissociation from the receptor. The active G protein leads to increased activity of cGMP phosphodiesterase and phosducin through its α and βγ subunits, respectively, and decreased guanylyl cyclase through its βγ subunit. In order for the photocycle to reset, the bound trans-retinal must dissociate from the receptor and be replaced with cis-retinol. For reverse opsins (B), trans-retinol can directly bind and activate the receptor, acting much like a pharmacological agonist. Light irradiation in this case leads to trans-cis isomerization, driving an inactive receptor conformation. In the case of OPNL1, bound cis-retinol is capable of thermally relaxing to the trans configuration, leading to receptor activation. For bistable opsins (C), one wavelength of light (in the case of parapinopsin, UV) induces cis-trans isomerization of the receptor-bound retinol, leading to receptor activation. Irradiation with a different wavelength of light (in this case, amber) is capable of inducing trans-cis isomerization, reverting the receptor back to its inactive form without necessitating retinol dissociation. For an Opto-XR chimeric receptor (D), specific C-terminal residues of a rhodopsin molecule are swapped for C-terminal residues of a nonvisual GPCR of interest. Light irradiation induces cis-trans retinol isomerization, activating the receptor. The active G protein drives increased or decreased adenylyl cyclase activity through the α subunit, depending on the selected GPCR chimera, increased GIRK conductance and PLCβ activity through the βγ, and decreased voltage-gated calcium channel (VGCC) conductance through the βγ subunit.

Fig. 2

Photopharmacology. (A) Ligand uncaging makes use of a photolabile protecting group (e.g., nitrobenzyl) conjugated to the compound of interest. After light (typically UV) cleavage of this group, the compound (in this case, enkephalin) is irreversibly liberated and able to interact with its cognate receptor. (B) Photoswitchable ligands (in this case Δ⁹-THC) have a photoisomerizable moiety (e.g., azobenzene) conjugated to them in a manner that allows for light-dependent conversion from an inactive to active form. After irradiation with a specific wavelength of light (γ₁), the photoswitchable group isomerizes (typically cis-trans) leading to an active form of the conjugated ligand, which can then bind to its cognate receptor. Irradiation with a different specific wavelength (γ₂) reverses the isomerization and reverts the compound back to its inactive form. (C) Tethered photoswitchable ligands rely on a rigid linker that attaches the ligand (in this case glutamate) to the photoisomerizable group to specific (typically cysteine) residues on the receptor itself. After irradiation with a specific wavelength of light (γ₁), the photoswitchable group isomerizes. In this case, the ligand is already in its active form, and the isomerization serves to either bring the ligand closer to the binding site or restrict it from the binding site. Note that the size and conformation of the linker are critically important in determining how the isomerization will ultimately effect ligand binding to the receptor. As with diffusible photoswitchable ligands, irradiation with a different specific wavelength (γ₂) reverses the isomerization. (D) Orthogonally tethered photoswitchable ligands (in this case 5-HT) are directly conjugated to the photoisomerizable moiety, similar to diffusible photoswitchable ligands. However, this photoisomerizable group is attached via a flexible linker that binds to a self-labeling tag (e.g., SNAP), which itself is conjugated to the receptor of interest, far from the orthosteric site. After irradiation with a specific wavelength of light (γ₁), the photoswitchable group isomerizes, leading to an active form of the conjugated ligand, which can then bind to its cognate receptor. Irradiation with a different specific wavelength (γ₂) reverses the isomerization and reverts the compound back to its inactive form.

Fig. 3

Optical control of intracellular signaling cascades. (A) Light-reactive protein domains such as BLUF undergo a conformational change when exposed to blue light. In nature, these domains are often directly conjugated to enzymes, such as adenylyl cyclase, such that the light-induced conformational change in the BLUF domain activates the associated enzyme (in this case, adenylyl cyclase). (B) AsLOV2 domains are a common strategy for light-dependent control of proteins or enzymes of interest. When fused to the LOV domain via the Jα helix, the protein is sequestered proximally to the LOV domain, sterically inhibiting it from interacting with other components of its signal transduction cascade. After light-dependent unwinding of the Jα helix, the now flexible linker allows the protein (in this case, Rac1) to diffuse away from LOV domain to interact with other protein partners (e.g., actin). (C) Many proteins, such as the RTKs or transcription factors, form functional homooligomers when activated. Light-dependent clustering schemes, such as those using the fungal photoreceptor VVD, have been used to gain optogenetic control over homooligomer formation (in this case, the transcription factor Gal4). (D) Alternatively, many studies have used light-evoked heterodimerization tools such as CRY2-C1B1 to gain control over specific protein-protein interactions. A light-induced conformational change in the CRY2 domain allows it to bind to its partner C1B1 and in doing so allows for conjugated proteins of interest to be brought in close proximity to each other (in this case, β-arrestin and the β₂AR).