Biased GPCR agonists that selectively engage G proteins or arrestins show therapeutic promise, but their development has been challenging. Key to understanding biased signaling is GRK2, which phosphorylates many GPCRs and facilitates arrestin recruitment. Canonically, GRK2 binds GPCRs only after being recruited to the plasma membrane by Gβγ, implying that arrestin recruitment requires prior G protein activation and challenging the feasibility of developing highly biased agonists. However, emerging evidence suggests that certain GPCRs, including dopamine D2 receptor, can bind GRK2 independently of Gβγ. Here, we combined a robust GRK2 recruitment assay and functional analyses to show that Gβγ-dependent and -independent GRK2 recruitment have distinct dynamics, signaling consequences, and impacts on bias. We further demonstrate that Gβγ-independent GRK2 recruitment facilitates arrestin recruitment to diverse GPCRs, revising the canonical model of GRK2 function. These findings highlight the need to account for distinct GRK2 recruitment mechanisms in the design of biased GPCR agonists.

Dopamine D1 receptor (D1R) plays key roles in health and disease. D1R is broadly expressed throughout the brain and body and is dynamically activated in response to endogenous dopamine, making it difficult to target this receptor with sufficient precision. We previously developed a robust light-activatable, tetherable agonist for D1R, wherein a temporally precise photo-switch (the P compound) binds to a genetically-encoded membrane anchoring protein (the M protein) in specific brain locations and cell types. Here we extended our approach by developing a complementary antagonist P compound that could be used to block specific populations of D1R in the brain with precise timing. Together, we have generated a robust toolkit for interrogating D1R function in the brain with unprecedented precision.

Timed dopamine signals underlie reinforcement learning, favoring neural activity patterns that drive behaviors with positive outcomes. In the striatum, dopamine activates five dopamine receptors (D1R-D5R), which are differentially expressed in striatal neurons. However, the role of specific dopamine receptors in reinforcement is poorly understood. Using our cell-specific D1R photo-agonist, we find that D1R activation in D1-expressing neurons in the dorsomedial striatum is sufficient to reinforce preceding neural firing patterns in defined ensembles of layer 5 cortico-striatal neurons of the mouse motor cortex. The reinforcement is cumulative and time dependent, with an optimal effect when D1R activation follows the selected neural pattern after a short interval. Our results show that D1R activation in striatal neurons can selectively reinforce cortical activity patterns, independent of a behavioral outcome or a reward, crucially contributing to the fundamental mechanisms that support cognitive functions like learning, memory, and decision-making.

AMPA receptors (AMPARs) are the main drivers of excitatory glutamatergic transmission in the brain, central to synaptic plasticity, and are key drug targets. However, AMPARs are expressed in virtually every neuron in the central nervous system and are activated with complex temporal dynamics, making it difficult to determine their functional roles with sufficient precision. Here we describe a cell specific, light-controllable competitive antagonist for the AMPA receptor called MP-GluAblock that combines the temporal precision of a photo-switchable ligand with the spatial and cellular specificity of a genetically-encoded membrane-anchor protein. This tool could pave the way for controlling endogenous AMPARs in neural circuits with cellular, spatial, and temporal specificity.

Dopamine D2-likereceptors (D2R, D3R, and D4R) control diversephysiological and behavioral functions and are important targets forthe treatment of a variety of neuropsychiatric disorders. Their complexdistribution and activation kinetics in the brain make it difficultto target specific receptor populations with sufficient precision.We describe a new toolkit of light-activatable, fast-relaxing, covalentlytaggable chemical photoswitches that fully activate, partially activate,or block D2-like receptors. This technology combines the spatiotemporalprecision of a photoswitchable ligand (P) with cell type and spatialspecificity of a genetically encoded membrane anchoring protein (M)to which the P tethers. These tools set the stage for targeting endogenousD2-like receptor signaling with molecular, cellular, and spatiotemporalprecision using only one wavelength of light.

G protein-coupled receptors (GPCRs) represent similar to 30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. (c) 2023 Wiley Periodicals LLC.Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptorsBasic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptorsAlternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membraneAlternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membraneSupport Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection

Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.

G protein-coupled receptors (GPCRs) are the most common targets of drug discovery. However, the similarity between related GPCRs combined with the complex spatiotemporal dynamics of receptor activation in vivo has hindered drug development. Photopharmacology offers the possibility of using light to control the location and timing of drug action by incorporating a photoisomerizable azobenzene into a GPCR ligand, enabling rapid and reversible switching between an inactive and active configuration. Recent advances in this area include (i) photoagonists and photoantagonists that directly control receptor activity but are nonselective because they bind conserved sites, and (ii) photoallosteric modulators that bind selectively to nonconserved sites but indirectly control receptor activity by modulating the response to endogenous ligand. In this study, we designed a photoswitchable allosteric agonist that targets a nonconserved allosteric site for selectivity and activates the receptor on its own to provide direct control. This work culminated in the development of aBINA, a photoswitchable allosteric agonist that selectively activates the Gi/o-coupled metabotropic glutamate receptor 2 (mGluR2). aBINA is the first example of a new class of precision drugs for GPCRs and other clinically important signaling proteins.

Dopamine controls diverse behaviors and their dysregulation contributes to many disorders. Our ability to understand and manipulate the function of dopamine is limited by the heterogenous nature of dopaminergic projections, the diversity of neurons that are regulated by dopamine, the varying distribution of the five dopamine receptors (DARs), and the complex dynamics of dopamine release. In order to improve our ability to specifically modulate distinct DARs, here we develop a photo-pharmacological strategy using a Membrane anchored Photoswitchable orthogonal remotely tethered agonist for the Dopamine receptor (MP-D). Our design selectively targets D1R/D5R receptor subtypes, most potently D1R (MP-D1 ago ), as shown in HEK293T cells. In vivo, we targeted dorsal striatal medium spiny neurons where the photo-activation of MP-D1 ago increased movement initiation, although further work is required to assess the effects of MP-D1 ago on neuronal function. Our method combines ligand and cell type-specificity with temporally precise and reversible activation of D1R to control specific aspects of movement. Our results provide a template for analyzing dopamine receptors.

Lysophosphatidic acid (LPA) is a phospholipid that acts as an extracellular signaling molecule and activates the family of lysophosphatidic acid receptors (LPA1-6). These G protein-coupled receptors (GPCRs) are broadly expressed and are particularly important in development as well as in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. Here, we report on a photoswitchable analogue of LPA, termed AzoLPA, which contains an azobenzene photoswitch embedded in the acyl chain. AzoLPA enables optical control of LPA receptor activation, shown through its ability to rapidly control LPA-evoked increases in intracellular Ca2+ levels. AzoLPA shows greater activation of LPA receptors in its light-induced cis-form than its dark-adapted (or 460 nm light-induced) trans-form. AzoLPA enabled the optical control of neurite retraction through its activation of the LPA2 receptor.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P(1-5)) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P(1-3), shown through electrophysiology and Ca2+ mobilization assays. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P(3)-dependent pain hypersensitivity in mice. The hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to the rapidly metabolized S1P. Using lipid mass spectrometry analysis, we constructed a metabolic map of PhotoS1P and PhotoSph. The formation of these photoswitchable lipids was found to be light dependent, providing a novel approach to optically probe sphingolipid biology.

Antipsychotic drugs (APDs) are some of the most widely used medications today. However, APDs also share prominent metabolic side effects, including weight gain, insulin resistance, and increased diabetes risk. The unifying property of all APDs is their blockade of dopamine D2-like receptors including D2 (D2R) and D3 receptors (D3R), suggesting the receptors' roles in metabolic disturbances. Besides brain expression, D2R and D3R are expressed in insulin-secreting pancreatic beta cells. Thus, we investigated D2R/D3R-mediated effects on glucose-stimulated insulin secretion (GSIS) and APD action directly in mouse pancreatic islets and INS-1E cells, a well-established rodent beta cell-derived cell line.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (membrane anchored Photoswitchable Orthogonal Remotely Tethered Ligand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic β-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic β-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in β-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in β-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which β-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, β-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson’s disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor’s downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in “indirect pathway” medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics.