G-protein-coupled receptors (GPCRs) are key mediators of cell communication and represent the most important class of drug targets1,2. Biophysical studies with purified GPCRs in vitro have suggested that they exist in an equilibrium of distinct inactive and active states, which is modulated by ligands in an efficacy-dependent manner3-11. However, how efficacy is encoded and whether multiple receptor states occur in living cells remain unclear. Here we use genetic code expansion12 and bioorthogonal labelling13-16 to generate a panel of fluorescence-based biosensors for a prototypical GPCR, the M2 muscarinic acetylcholine receptor (M2R). These biosensors enable real-time monitoring of agonist-promoted conformational changes across the receptor's extracellular surface in intact cells. We demonstrate that different agonists produce equilibria of at least four distinct active states of the G-protein-bound M2R, each with a different ability to activate G proteins. The formation of these M2R-G-protein complexes occurs over 0.2-5 s along trajectories that involve both common and ligand-specific conformational changes and appear to determine G-protein selectivity. These observations reveal the molecular nature of ligand efficacy in intact cells. Selectively exploiting such different GPCR activation trajectories and conformational equilibria may open new avenues for GPCR drug discovery.

G-protein-coupled receptors (GPCRs) are the largest class of membrane receptors and key drug targets. Over the past decade, extensive evidence has shown that GPCRs signal from various intracellular compartments to generate distinct cellular and physiological responses. Therefore, it is critical to understand how the subcellular sites of GPCR signaling affect receptor function and pharmacology, and how the insights can be exploited to develop improved therapeutics. This Review highlights recent advances in GPCR signaling and spatiotemporal regulation with a focus on chemical biology approaches. We discuss biosensors for real-time recordings of localized GPCR responses, tools for site-selective tuning of signaling pathways, quantitative approaches to determine interactors and global cellular behaviors, reporters enabling unbiased mechanistic screening and compartment-specific ligands. We emphasize the need to develop and advance chemical biology tools to deepen our mechanistic understanding of compartmentalized GPCR signaling and fuel future drug discovery efforts.

In a study published recently in Cell,Motso et al.1 developed a series of G protein-coupled receptor kinase 2(GRK2)-biased agonists for the β2-adrenergic receptor(β2AR)which showed efficacy in stimulating muscular glucose uptake without eliciting cardiac side effects typically associated with systemically appliedβ-agonists.2 The candidate drug,compound 15,was well-tolerated in a phase 1 clinical trial and could provide a promising alternative treatment option for type 2 diabetes and obesity.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64–65. ADAM-19 cleavage increased G q and decreased G s activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

Louis Pasteur once famously said 'in the fields of observation chance favors only the prepared mind'. Much of chance is being in the right place at the right time. This is particularly true in the crowded molecular environment of the cell where being in the right place is often more important than timing. Although Brownian motion argues that enzymes will eventually bump into substrates, this probability is greatly enhanced if both molecules reside in the same subcellular compartment. However, activation of cell signaling enzymes often requires the transmission of chemical signals from extracellular stimuli to intracellular sites of action. This review highlights new developments in our understanding of cAMP generation and the 3D utilization of this second messenger inside cells.

G protein-coupled receptors are the largest and pharmacologically most important receptor family and are involved in the regulation of most cell functions. Most of them reside exclusively at the cell surface, from where they signal via heterotrimeric G proteins to control the production of second messengers such as cAMP and IP3 as well as the activity of several ion channels. However, they may also internalize upon agonist stimulation or constitutively reside in various intracellular locations. Recent evidence indicates that their function differs depending on their precise cellular localization. This is because the signals they produce, notably cAMP and Ca2+, are mostly bound to cell proteins that significantly reduce their mobility, allowing the generation of steep concentration gradients. As a result, signals generated by the receptors remain confined to nanometer-sized domains. We propose that such nanometer-sized domains represent the basic signaling units in a cell and a new type of target for drug development.

G protein-coupled receptors (GPCRs) control cell functions by responding to a myriad of extracellular signals, such as hormones and neurotransmitters. GPCRs comprise the most important drug targets. Cells express many different GPCRs each eliciting distinct and specific cell functions; however, GPCRs use only a few second messengers such as cyclic adenosine monophosphate (cAMP) to relay precise receptor stimuli. To control cell signaling specificity, we showed recently that cells organize subcellular cAMP signaling in so-called cAMP nanodomains, namely nanometer-sized signaling compartments of different local cAMP concentrations. GPCRs orchestrate high-concentration cAMP nanodomains to stimulate cell signaling, whereas phosphodiesterases (PDEs), enzymes that degrade cAMP, control low-concentration cAMP nanodomains to gate cAMP effector activation and downstream signaling. This precise cAMP signaling nanoarchitecture is essential for cell homeostasis. HTNB is a Mendelian hypertension form that results in dramatic increases in blood pressure leading to death by stroke at the age of 50 years when untreated. Increased peripheral vascular resistance is responsible. We present an HTNB patient with a novel, single-point mutation (L910P) in the catalytic PDE3A core. The localization indicates a direct influence on cAMP hydrolysis. We developed a FRET-based in vitro assay to determine the hydrolytic activities of the disease-causing PDE3 mutant to be 3-fold higher than PDE3A wt (V max (nM (cAMP)/s) were 143±7 and 45±3, respectively), substantially greater than earlier mutants we described. We used our recently developed nanoruler imaging technology for cAMP and downstream effectors and found that the increased cAMP turnover leads to pathologically large low-cAMP nanodomains in vicinity of PDE3A, thereby altering downstream cAMP signaling. Our data suggest that altered cAMP signaling, at the nanometer scale, causes the hypercontractile vascular smooth muscle phenotype and increased peripheral resistance in HTNB, since local protein kinase-A (PKA) activity is hypoactive in the PDE vicinity. We propose that disrupted cellular cAMP-signaling nanoarchitecture forms the molecular basis for HTNB. - 1 -

G protein-coupled receptors (GPCRs) control cell functions by responding to a myriad of extracellular signals, such as hormones and neurotransmitters. GPCRs comprise the most important drug targets. Cells express many different GPCRs each eliciting distinct and specific cell functions; however, GPCRs use only a few second messengers such as cyclic adenosine monophosphate (cAMP) to relay precise receptor stimuli. To control cell signaling specificity, we showed recently that cells organize subcellular cAMP signaling in so-called cAMP nanodomains, namely nanometer-sized signaling compartments of different local cAMP concentrations. GPCRs orchestrate high-concentration cAMP nanodomains to stimulate cell signaling, whereas phosphodiesterases (PDEs), enzymes that degrade cAMP, control low-concentration cAMP nanodomains to gate cAMP effector activation and downstream signaling. This precise cAMP signaling nanoarchitecture is essential for cell homeostasis. HTNB is a Mendelian hypertension form that results in dramatic increases in blood pressure leading to death by stroke at the age of 50 years when untreated. Increased peripheral vascular resistance is responsible. We present an HTNB patient with a novel, single-point mutation (L910P) in the catalytic PDE3A core. The localization indicates a direct influence on cAMP hydrolysis. We developed a FRET-based in vitro assay to determine the hydrolytic activities of the disease-causing PDE3 mutant to be 3-fold higher than PDE3A wt (V max (nM (cAMP)/s) were 143±7 and 45±3, respectively), substantially greater than earlier mutants we described. We used our recently developed nanoruler imaging technology for cAMP and downstream effectors and found that the increased cAMP turnover leads to pathologically large low-cAMP nanodomains in vicinity of PDE3A, thereby altering downstream cAMP signaling. Our data suggest that altered cAMP signaling, at the nanometer scale, causes the hypercontractile vascular smooth muscle phenotype and increased peripheral resistance in HTNB, since local protein kinase-A (PKA) activity is hypoactive in the PDE vicinity. We propose that disrupted cellular cAMP-signaling nanoarchitecture forms the molecular basis for HTNB. - 1 -

G protein-coupled receptors (GPCRs) are ligand-activated cell membrane proteins and represent the most important class of drug targets. GPCRs adopt several active conformations that stimulate different intracellular G proteins (and other transducers) and thereby modulate second messenger levels, eventually resulting in receptor-specific cell responses. It is increasingly accepted that not only the type of active signaling protein but also the duration of its stimulation and the subcellular location from where receptors signal distinctly contribute to the overall cell response. However, the molecular principles governing such spatiotemporal GPCR signaling and their role in disease are incompletely understood. Genetically encoded, fluorescent biosensors-in particular for the GPCR/cAMP signaling axis-have been pivotal to the discovery and molecular understanding of novel concepts in spatiotemporal GPCR signaling. These include GPCR priming, location bias, and receptor-associated independent cAMP nanodomains. Here, we review such technologies that we believe will illuminate the spatiotemporal organization of other GPCR signaling pathways that define the complex signaling architecture of the cell.

The exact interplay between receptors and their downstream signaling influences essentially all physiological functions. But how can a cell discriminate between hundreds of different receptors that share the same downstream signaling transducers? We find receptor-associated independent signaling nanodomains (RAINs) around single receptors which can specifically switch signaling cascades on or off.

G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and β2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.

3',5'-cyclic adenosine monophosphate (cAMP) is one of the most important and ubiquitous second messengers in cells downstream of G protein-coupled receptors (GPCRs). In a single cell, cAMP can exert innumerous specific cell functions in response to more than one hundred different GPCRs. Cells achieve this extraordinary functional specificity of cAMP signaling by limiting the spread of these signals in space and time. To do so, cells establish nanometer-size cAMP gradients by immobilizing cAMP via cAMP binding proteins and via targeted activity of cAMP-degrading phosphodiesterases (PDEs). As cAMP gradients appear to be essential for cell function, new technologies are needed to accurately measure cAMP gradients in intact cells with nanometer-resolution. Here we describe FRET-based cAMP nanorulers to measure local, nanometer-size cAMP gradients in intact cells in the direct vicinity of PDEs.

Mendelian syndromes give great insight into pathogenesis and have implicated salt handling. Hypertension with brachydactyly (HTNB) is unique in that a direct increase in peripheral vascular resistance is produced by activating mutations in phosphodiesterase 3A (PDE3A). A 50 mm Hg blood-pressure elevation by age 50 years causes stroke in untreated persons. Here, we report mutations in the PDE3 catalytic domain found in two new HTNB families. Since structural predictions indicated increased PDE3A cleavage activity, we used Förster resonance energy transfer (FRET) to measure the PDE3-specific cAMP degradation in cytosolic fractions from transfected cells. The newly discovered catalytic-domain PDE3A mutants, R862C and L910P, both showed a shorter transient emission-ratio change upon cAMP addition than PDE3A wildtype, indicating faster cAMP turnover. The V max of all experiments were extracted from FRET data and quantitated and analyzed statistically. The mean values V max (nM (cAMP)/s) were 143±7 for L910P, 63±3 for R862C and 73±4 for T445N, and 45±3 for WT respectively, (p<0.0001, <0.001, <0.05). We found that all disease-relevant mutations led to a clearly hyperactive PDE3A. The L910P catalytic-domain mutation resulted in the most dramatic increase in cAMP hydrolysis. That mutation also exhibited the most stepwise resistance to milrinone. Recently, we showed that cAMP signaling is organized in nanometer-sized cellular compartments. We suggest that the gain-of-function PDE3 mutations result in unphysiologically larger cAMP nanodomains and thereby dysregulate crucially important cAMP nanoarchitecture. Spatiotemporal cAMP signaling and precise PDE-controlled effector activation, could usher in a site-specific nanomolecular pharmacology.

Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

In July 2021, we organized a virtual symposium aimed at early-career investigators (ECIs) in G protein-coupled receptor (GPCR) research: the first Transatlantic ECI GPCR Symposium. Here, we discuss the proceedings of this symposium and the unique networking events with GPCR leaders including the Nobel Laureates Dr. Robert Lefkowitz and Dr. Brian Kobilka.

Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.

G protein‐coupled receptors (GPCRs) are essential cell membrane signaling molecules and represent the most important class of drug targets. Some signaling pathways downstream of a GPCR may be responsible for drug adverse effects, while others mediate therapeutic efficacy. Biased ligands preferentially activate only a subset of all GPCR signaling pathways. They hold great potential to become next‐generation GPCR drugs with less side effects due to their potential to exclusively activate desired signaling pathways. However, the molecular basis of biased agonism is poorly understood. GPCR activation occurs through allosteric coupling, the propagation of conformational changes from the extracellular ligand‐binding pocket to the intracellular G protein‐binding interface. Comparison of GPCR structures in complex with G proteins or β‐arrestin reveals that intracellular transducer coupling results in closure of the ligand‐binding pocket trapping the agonist inside its binding site. Allosteric coupling appears to be transducer‐specific offering the possibility of harnessing this mechanism for the design of biased ligands. Here, we review the biochemical, pharmacological, structural, and biophysical evidence for allosteric coupling and delineate that biased agonism should be a consequence of preferential allosteric coupling from the ligand‐binding pocket to one transducer‐binding site. As transducer binding leads to large structural rearrangements in the extracellular ligand‐binding pocket, we survey biased ligands with an extended binding mode that interact with extracellular receptor domains. We propose that biased ligands use ligand‐specific triggers inside the binding pocket that are relayed through preferential allosteric coupling to a specific transducer, eventually leading to biased signaling.