cAMP signaling: a remarkably regional affair.

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.

Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

Selective activation of intracellular ß1AR using a spatially restricted antagonist.

G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for ß1-adrenergic receptor (ß1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable ß1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited ß1AR on the plasma membrane. In contrast, a cell permeable ß1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of ß1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while ß1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized ß1AR signaling in various context, potentially advancing our understanding of ß1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus.

Dopamine is a key catecholamine in the brain and kidney, where it is involved in a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation, and renal function. As a membrane-impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein coupled receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on organic cation transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane-impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in murine striatal medium spiny neurons, and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gαs coupling using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data provide further evidence for the role of transporters in regulating subcellular GPCR activity.

G protein-regulated endocytic trafficking of adenylyl cyclase type 9.

GPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report selective trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs. AC9 transits a similar, dynamin-dependent early endocytic pathway as ligand-activated GPCRs. However, unlike GPCR traffic control which requires ß-arrestin but not Gs, AC9 traffic control requires Gs but not ß-arrestin. We also show that AC9, but not AC1, mediates cAMP production stimulated by endogenous receptor activation in endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in regulating the subcellular distribution of a relevant effector.

Cells sense changes in their chemical environment using proteins called receptors. These proteins often sit on the cell surface, detecting molecules outside the cell and relaying messages across the membrane to the cell interior. The largest family of receptors is formed of 'G protein-coupled receptors' (or GPCRs for short), so named because they relay messages through so-called G proteins, which then send information into the cell by interacting with other proteins called effectors. Next, the receptors leave the cell surface, travelling into the cell in compartments called endosomes. Researchers used to think that this switched the receptors off, stopping the signaling process, but it is now clear that this is not the case. Some receptors continue to signal from inside the cell, though the details of how this works are unclear. For signals to pass from a GPCR to a G protein to an effector, all three proteins need to be in the same place. This is certainly happening at the cell surface, but whether all three types of proteins come together inside endosomes is less clear. One way to find out is to look closely at the location of effector proteins when GPCRs are receiving signals. One well-studied effector of GPCR signaling is called adenylyl cyclase, a protein that makes a signal molecule called cAMP. Some G proteins switch adenylyl cyclase on, increasing cAMP production, while others switch it off. To find out how GPCRs send signals from inside endosomes, Lazar et al tracked adenylyl cyclase proteins inside human cells. This revealed that a type of adenylyl cyclase, known as adenylyl cyclase 9, follows receptors as they travel into the cell. Under the influence of active G proteins, activated adenylyl cyclase 9 left the cell surface and entered the endosomes. Once inside the cell, adenylyl cyclase 9 generated the signal molecule cAMP, allowing the receptors to send messages from inside the cell. Other types of adenylyl cyclase behaved differently. Adenylyl cyclase 1, for example, remained on the cell surface even after its receptors had left, and did not signal from inside the cell at all. Which cell behaviors are triggered from the membrane, and which are triggered from inside the cell is an important question in drug design. Understanding where effector proteins are active is a step towards finding the answers. This could help research into diseases of the heart, the liver and the lungs, all of which use adenylyl cyclase 9 to send signals.

Emerging Role of Compartmentalized G Protein-Coupled Receptor Signaling in the Cardiovascular Field.

G protein-coupled receptors (GPCRs) are cell surface receptors that for many years have been considered to function exclusively at the plasma membrane, where they bind to extracellular ligands and activate G protein signaling cascades. According to the conventional model, these signaling events are rapidly terminated by ß-arrestin (ß-arr) recruitment to the activated GPCR resulting in signal desensitization and receptor internalization. However, during the past decade, emerging evidence suggest that many GPCRs can continue to activate G proteins from intracellular compartments after they have been internalized. G protein signaling from intracellular compartments is in general more sustained compared to G protein signaling at the plasma membrane. Notably, the particular location closer to the nucleus is beneficial for selective cellular functions such as regulation of gene transcription. Here, we review key GPCRs that undergo compartmentalized G protein signaling and discuss molecular considerations and requirements for this signaling to occur. Our main focus will be on receptors involved in the regulation of important physiological and pathological cardiovascular functions. We also discuss how sustained G protein activation from intracellular compartments may be involved in cellular functions that are distinct from functions regulated by plasma membrane G protein signaling, and the corresponding significance in cardiovascular physiology.

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.

Golgi localized ß1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of ß1 adrenergic receptors (ßARs) on cardiac myocytes. Blocking these receptors is part of the basis for ß-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including ßARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi ßARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface ßAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident ß1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious ß-blocker therapies.

Genetically Encoding Quinoline Reverses Chromophore Charge and Enables Fluorescent Protein Brightening in Acidic Vesicles.

Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally nonfluorescent at acidic conditions due to the decrease of anionic chromophores upon protonation, but are fluorescent at physiological pH, creating interfering fluorescence from nonvesicle regions. Here we developed a novel acid-brightening fluorescent protein (abFP) that fluoresces strongly at acidic pH but is nonfluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on the mammalian cell surface. We further demonstrated that abFP-tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP-tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.

Functional selectivity of GPCR-directed drug action through location bias.

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The ß2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human ß1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that ß-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.

Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

Retromer mediates sequence-directed cargo exit from endosomes to support both endosome-to-Golgi (retrograde transport) and endosome-to-plasma membrane (recycling) itineraries. It is not known whether these trafficking functions require cargos to exit endosomes separately via distinct transport intermediates or whether the same retromer-coated carriers can support both itineraries. We addressed this question by comparing human Wntless (Wls) and ß2 adrenergic receptor (ß2AR), which require retromer physiologically for retrograde transport and recycling, respectively. We show here by direct visualization in living cells that both cargos transit primarily the same endosomes and exit via shared transport vesicles generated from a retromer-coated endosome domain. While both Wls and ß2AR clearly localize to the same retromer-coated endosome domains, Wls is consistently enriched more strongly. This enrichment difference is determined by distinct motifs present in the cytoplasmic tail of each cargo, with Wls using tandem Φ-X-[L/M] motifs and ß2AR using a PDZ motif. Exchanging these determinants reverses the enrichment phenotype of each cargo but does not change cargo itinerary, verifying the multifunctional nature of retromer and implying that additional sorting must occur downstream. Quantitative differences in the degree of cargo enrichment instead underlie a form of kinetic sorting that impacts the rate of cargo delivery via both itineraries and determines the ability of ß2AR to activate its cognate G protein transducer locally from endosomes. We propose that mammalian retromer forms a multifunctional membrane coat that supports shared cargo exit for divergent trafficking itineraries and regulates signaling from endosomes.

The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the ß2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/ß-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the ß2-adrenergic receptor (ß2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the ß2AR in a ligand-independent manner. Although ARRDC3 has no effect on ß2AR endocytosis or degradation, it negatively regulates ß2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in ß2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the ß2AR.

Effects of endocytosis on receptor-mediated signaling.

Cellular mechanisms of membrane traffic and signal transduction are deeply interconnected. The present review discusses how membrane trafficking in the endocytic pathway impacts receptor-mediated signaling. Examples of recent progress are highlighted, focusing on the endocytosis-signaling nexus in mammals.

G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors.

GPCR signaling along the endocytic pathway.

Many G protein-coupled receptors (GPCRs) internalize after agonist-induced activation. While endocytosis has long been associated with homeostatic attenuation of cellular responsiveness, accumulating evidence from study of a wide range of eukaryotes reveals that the endocytic pathway also contributes to generating receptor-initiated signals themselves. Here we review recent progress in this area, discussing primarily but not exclusively GPCR signaling in mammalian cells.

Investigating signaling consequences of GPCR trafficking in the endocytic pathway.

Ligand-dependent regulation of adenylyl cyclase by the large family of seven-transmembrane G protein-coupled receptors (GPCRs) represents a deeply conserved and widely deployed cellular signaling mechanism. Studies of adenylyl cyclase regulation by catecholamine receptors have led to a remarkably detailed understanding of the basic biochemistry of G protein-linked signal transduction and have elaborated numerous mechanisms of regulation. Endocytosis of GPCRs plays a significant role in controlling longer-term cellular responses, such as under conditions of prolonged or repeated receptor activation occurring over a course of hours or more. It has been more challenging to investigate regulatory effects occurring over shorter time intervals, within the minutes to tens of minutes spanning the time course of many acute cyclic AMP (cAMP)-mediated signaling processes. A main reason for this is that biochemical methods used traditionally to assay changes in cytoplasmic cAMP concentration are limited in spatiotemporal resolution and typically require perturbing cellular structure and/or function for implementation. Recent developments in engineering genetically encoded cAMP biosensors linked to optical readouts, which can be expressed in cells or tissues and detected without cellular disruption or major functional perturbation, represent a significant step toward overcoming these limitations. Here, we describe the application of two such cAMP biosensors, one based on enzyme complementation and luminescence detection and another using Förster resonance energy transfer and fluorescence detection. We focus on applying these approaches to investigate cAMP signaling by catecholamine receptors and then on combining these analytical approaches with manipulations of receptor endocytic trafficking.

Conformational biosensors reveal GPCR signalling from endosomes.

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the ß2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

Regulation of constitutive cargo transport from the trans-Golgi network to plasma membrane by Golgi-localized G protein betagamma subunits.

Observations of Golgi fragmentation upon introduction of G protein ßγ (Gßγ) subunits into cells have implicated Gßγ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gßγ acts to provoke Golgi fragmentation is not known. Additionally, a role for Gßγ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gßγ to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted ß(1)γ(2) also activates protein kinase D. Moreover, the novel Gßγ inhibitor, gallein, and the Gßγ-sequestering protein, GRK2ct, reveal that Gßγ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gßγ, endogenous Gß was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized Gßγ in regulating protein transport from the TGN to the cell surface.

Assembly and trafficking of heterotrimeric G proteins.

To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.