Prototypical G Protein-coupled Receptors Research Articles

Sodium ions allosterically modulate the M2 muscarinic receptor

G protein coupled receptors (GPCRs) play a key role in the vast majority of cellular signal transduction processes. Previous experimental evidence has shown that sodium ion (Na+) allosterically modulate several class A GPCRs and theoretical studies suggested that the same also holds true for muscarinic receptors. Here we examined, using Xenopus oocytes as an expression system, the effect of Na+ on a prototypical GPCR, the M2 muscarinic receptor (M2R). We found that removal of extracellular Na+ resulted in a decrease in the potency of ACh toward the M2R and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. We further show that this allosteric effect of Na+ does not underlie the voltage-dependence of this receptor.

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.

TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome.

G protein-coupled receptors (GPCRs) remain major drug targets despite our incomplete understanding of how they signal through 16 non-visual G protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we developed an open-source suite of 14 optimized Bioluminescence Resonance Energy Transfer (BRET) Gαβγ biosensors (dubbed TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.

Exploring Biased Agonism at FPR1 as a Means to Encode Danger Sensing.

Ligand-based selectivity in signal transduction (biased signaling) is an emerging field of G protein-coupled receptor (GPCR) research and might allow the development of drugs with targeted activation profiles. Human formyl peptide receptor 1 (FPR1) is a GPCR that detects potentially hazardous states characterized by the appearance of N-formylated peptides that originate from either bacteria or mitochondria during tissue destruction; however, the receptor also responds to several non-formylated agonists from various sources. We hypothesized that an additional layer of FPR signaling is encoded by biased agonism, thus allowing the discrimination of the source of threat. We resorted to the comparative analysis of FPR1 agonist-evoked responses across three prototypical GPCR signaling pathways, i.e., the inhibition of cAMP formation, receptor internalization, and ERK activation, and analyzed cellular responses elicited by several bacteria- and mitochondria-derived ligands. We also included the anti-inflammatory annexinA1 peptide Ac2-26 and two synthetic ligands, the W-peptide and the small molecule FPRA14. Compared to the endogenous agonists, the bacterial agonists displayed significantly higher potencies and efficacies. Selective pathway activation was not observed, as both groups were similarly biased towards the inhibition of cAMP formation. The general agonist bias in FPR1 signaling suggests a source-independent pathway selectivity for transmission of pro-inflammatory danger signaling.

Dissection of anterograde trafficking and sorting of GPCRs in primary neurons

G protein‐coupled receptors (GPCRs) play an important role in regulating the function of the nervous system. However, the molecular mechanisms underlying the targeting and sorting of GPCRs to the functional destination in neurons are not well understood. Here we investigated the role of small GTPase Rab43, via dominant negative inhibition and CRISPR‐Cas9‐mediated knockout, in the transport of several prototypic GPCRs to dendrites and spines and elucidated the possible mechanisms. We demonstrated that inhibition of Rab43 function significantly reduced the surface numbers of α2‐adrenergic receptors (ARs) and β2‐ARs, but not M3‐muscarinic receptors (MRs) in primary cultures of neurons as measured by live neuron radioligand binding. Confocal microscopy revealed that three receptors utilized similar structural determinants, including the highly conserved LL motif in the C‐termini, for their anterograde transport, and that Rab43 affected the expression of α2‐ARs, but not M3‐MRs and vesicular stomatitis virus glycoprotein, at both dendrites and spines. Interestingly, Rab43 only altered β2‐AR expression at spines, but not dendrites. We also found that Rab43 directly and activation‐dependently interacted with α2‐ARs, but not M3‐MRs, and the Rab43‐binding domain was located in the third intracellular loop of the receptor. The chimeric M3‐MRs containing the Rab43‐binding domain of α2‐ARs were subject to Rab43 regulation. These data demonstrate distinct, as yet unidentified pathways that transport different GPCRs to dendrites and spines, in which Rab43 plays a crucial role in the sorting and trafficking of the receptors likely via direct interaction.Support or Funding InformationNIH R01 GM118915

G-Protein-Coupled Receptor-Membrane Interactions Depend on the Receptor Activation State.

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A1 receptor (A1 AR) is an important therapeutic target for treating cardiac ischemia-reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A1 AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid-protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A1 AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A1 AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A1 AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.

A Naturally Occurring Splice Variant of GGA1 Inhibits the Anterograde Post-Golgi Traffic of \u03b12B-Adrenergic Receptor

The regulatory mechanisms of cell surface targeting of nascent G protein-coupled receptors (GPCRs) en route from the endoplasmic reticulum through the Golgi remain poorly understood. We have recently demonstrated that three Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins (GGAs) mediate the post-Golgi export of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and directly interact with the receptor. In particular, GGA1 interaction with α2B-AR is mediated via its hinge domain. Here we determined the role of a naturally occurring truncated form of GGA1 (GGA1t) which lacks the N-terminal portion of the hinge domain in α2B-AR trafficking and elucidated the underlying mechanisms. We demonstrated that both GGA1 and GGA1t were colocalized and mainly expressed at the Golgi. In marked contrast to GGA1, the expression of GGA1t significantly attenuated the cell surface export of newly synthesized α2B-AR from the Golgi and in parallel receptor-mediated signaling. Furthermore, we found that GGA1t formed homodimers and heterodimers with GGA1. More interestingly, GGA1t was unable to bind the cargo α2B-AR and to recruit clathrin onto the trans-Golgi network. These data provide evidence implicating that the truncated form of GGA1 behaviors as a dominant-negative regulator for the cell surface export of α2B-AR and this function of GGA1t is attributed to its abilities to dimerize with its wide type counterpart and to inhibit cargo interaction and clathrin recruitment to form specialized transport vesicles.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes.

Membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. Force-distance curve-based AFM (FD-AFM) was utilized to directly probe and localize the conformational states of a GPCR within the membrane at nanoscale resolution based on the mechanical properties of the receptor. FD-AFM was applied to rhodopsin, the light receptor and a prototypical GPCR, embedded in native rod outer segment disc membranes from photoreceptor cells of the retina in mice. Both FD-AFM and computational studies on coarse-grained models of rhodopsin revealed that the active state of the receptor has a higher Young's modulus compared to the inactive state of the receptor. Thus, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Differentiating the states based on the Young's modulus allowed for the mapping of the different states within the membrane. Quantifying the active states present in the membrane containing the constitutively active G90D rhodopsin mutant or apoprotein opsin revealed that most receptors adopt an active state. Traditionally, constitutive activity of GPCRs has been described in terms of two-state models where the receptor can achieve only a single active state. FD-AFM data are inconsistent with a two-state model but instead require models that incorporate multiple active states.

β2-Adrenergic Stimulation Compartmentalizes β1 Signaling Into Nanoscale Local Domains by Targeting the C-Terminus of β1-Adrenoceptors.

βARs (β-adrenergic receptors) are prototypical GPCRs (G protein-coupled receptors) that play a pivotal role in sympathetic regulation. In heart cells, β1AR signaling mediates a global response, including both l-type Ca2+ channels in the sarcolemma/T tubules and RyRs (ryanodine receptors) in the SR (sarcoplasmic reticulum). In contrast, β2AR mediates local signaling with little effect on the function of SR proteins. To investigate the signaling relationship between β1ARs and β2ARs. Using whole-cell patch-clamp analyses combined with confocal Ca2+ imaging, we found that the activation of compartmentalized β2AR signaling was able to convert the β1AR signaling from global to local mode, preventing β1ARs from phosphorylating RyRs that were only nanometers away from sarcolemma/T tubules. This offside compartmentalization was eliminated by selective inhibition of β2AR, GRK2 (GPCR kinase-2), βarr1 (β-arrestin-1), and phosphodiesterase-4. A knockin rat model harboring mutations of the last 3 serine residues of the β1AR C terminus, a component of the putative βarr1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by β2AR activation. β2AR stimulation compartmentalizes β1AR signaling into nanoscale local domains in a phosphodiesterase-4-dependent manner by targeting the C terminus of β1ARs. This finding reveals a fundamental negative feed-forward mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient β1AR response of sympathetic excitation.

Specific TBC Domain‐Containing Proteins Control the ER‐Golgi‐Plasma Membrane Trafficking of GPCRs

G protein‐coupled receptors (GPCRs) constitute the largest superfamily of cell‐surface signaling proteins. However, the molecular mechanisms underlying their cell surface delivery after synthesis remain poorly understood. Here we screen the TBC domain‐containing proteins, putative Rab GTPase‐activating proteins (GAPs), in the intracellular trafficking of GPCRs and identify several TBC proteins that activity‐dependently regulate the anterograde transport, en route from the endoplasmic reticulum to the Golgi or from the Golgi to the cell surface, of several prototypic GPCR members without affecting other plasma membrane proteins. We also show that TBC1D6 functions as a GAP for Rab26, physically associates with Rab26, and attenuates Rab26 interaction with GPCRs. Furthermore, both overexpression and depletion of TBC1D6 inhibit the post‐Golgi traffic of GPCRs. These data demonstrate for the first time the important roles of the TBC proteins in forward trafficking of nascent GPCRs and reveal novel regulatory mechanisms of GPCR targeting to the functional destination.Support or Funding InformationNIH R01GM118915This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein–coupled receptors (GPCRs) called somatostatin receptor (SST)1–5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Molecular details of dimerization kinetics reveal negligible populations of transient \xb5-opioid receptor homodimers at physiological concentrations

Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization.

Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of β2-adrenergic receptor (β2AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric β2ARs that undergo endocytosis, whereas PKA modifies dimeric β2ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated β2ARs are enriched in dendrites, whereas GRK-phosphorylated β2ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated β2ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell.

Identification of GPCR Transition Pathways using Go Models

One caveat of standard all-atom molecular dynamics (MD) simulations is that they are resource-intensive, particularly when inspecting state transition pathways. To date, the timescales involved in processes such as G protein-coupled receptor (GPCR) activation make it largely impossible to study them using standard all-atom MD under equilibrium conditions. Indeed, the nature of their activation/deactivation pathway(s) is not yet fully characterized. To overcome this issue, our group previously showed that simpler structure-based (Go-like) potentials could be used to study GPCR state transitions, yielding results in qualitative agreement with those obtained from standard all-atom simulations but with negligible computational cost. These potentials are energetically smooth by design with few non-native traps and a single global energy minimum corresponding to the end-point of the transition of interest. In the case of rhodopsin, a prototypical GPCR, two distinct but reproducible pathways were observed to be populated during activation and deactivation. While it is possible that a separation of pathways is mechanistically required for function, it could also be the result of simulating state transitions occurring in two separate energy surfaces out of equilibrium (one for activation and one for deactivation). To address this potential source of error, we implemented a strategy for modeling Go-like energy landscapes with multiple energy minima at all-heavy atom resolution. This approach allows the study of state interconversion in equilibrium and extensive sampling, still at a low computational cost. Moreover, it can also be used for quick hypothesis testing, to characterize the effects of different ligands, model point mutations, study substate fluctuations and guide all-atom simulations by quickly generating reasonable reaction coordinates. As long as the end states are known, this strategy can be extrapolated to almost any biomolecular system in a straight forward manner, complementing current standard all-atom MD approaches.

Characterizing GPCR Allostery by NMR Spectroscopy

Forty percent of current pharmaceuticals target G protein-coupled receptors (GPCRs), a class of membrane proteins that regulate diverse biological processes including sensory perception, hormonal regulation, and immune response. Structurally, GPCRs exist in a dynamic ensemble of conformations spanning inactive, intermediate, and active states capable of engaging G proteins. The adenosine A2A receptor (A2AR) is a prototypical GPCR and a drug target for the treatment of inflammation, cancer, diabetes, and Parkinson's disease. While X-ray crystal structures reveal only an inactive and an active conformation, nuclear magnetic resonance (NMR) spectroscopy show that detergent-reconstituted A2AR exhibits at least four functional states spanning inactive, intermediate, and active states capable of engaging G proteins. Though mechanistically poorly understood, the lipid bilayer plays an important role in GPCR regulation. Several GPCRs are known to require membrane cholesterol for function, including A2AR. Here, we employ fluorine (19F) NMR to characterize A2AR activity and drug-response in a synthetic lipid bilayer (nanodisc). We show that cholesterol is an allosteric effector of A2AR through modulation of its functional states. This knowledge furthers our understanding of GPCR allostery and protein-membrane interactions.

Exploring a new ligand binding site of G protein-coupled receptors.

Identifying a target ligand binding site is an important step for structure-based rational drug design as shown here for G protein-coupled receptors (GPCRs), which are among the most popular drug targets. We applied long-time scale molecular dynamics simulations, coupled with mutagenesis studies, to two prototypical GPCRs, the M3 and M4 muscarinic acetylcholine receptors. Our results indicate that unlike synthetic antagonists, which bind to the classic orthosteric site, the endogenous agonist acetylcholine is able to diffuse into a much deeper binding pocket. We also discovered that the most recently resolved crystal structure of the LTB4 receptor comprised a bound inverse agonist, which extended its benzamidine moiety to the same binding pocket discovered in this work. Analysis on all resolved GPCR crystal structures indicated that this new pocket could exist in most receptors. Our findings provide new opportunities for GPCR drug discovery.

The orientation and stability of the GPCR-Arrestin complex in a lipid bilayer

G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that plays a key role in transmembrane signal transduction and draw wide attention since it was discovered. Arrestin is a small family of proteins which can bind to GPCRs, block G protein interactions and redirect signaling to G-protein-independent pathways. The detailed mechanism of how arrestin interacts with GPCR remains elusive. Here, we conducted molecular dynamics simulations with coarse-grained (CG) and all-atom (AA) models to study the complex structure formed by arrestin and rhodopsin, a prototypical GPCR, in a POPC bilayer. Our results indicate that the formation of the complex has a significant impact on arrestin which is tightly anchored onto the bilayer surface, while has a minor effect on the orientation of rhodopsin in the lipid bilayer. The formation of the complex induces an internal change of conformation and flexibility in both rhodopsin and arrestin, mainly at the binding interface. Further investigation on the interaction interface identified the hydrogen bond network, especially the long-lived hydrogen bonds, and the key residues at the contact interface, which are responsible for stabilizing the complex. These results help us to better understand how rhodopsin interacts with arrestin on membranes, and thereby shed lights on arrestin-mediated signal transduction through GPCRs.

Integration of GPCR Signaling and Sorting from Very Early Endosomes via Opposing APPL1 Mechanisms

SummaryEndocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized β2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.

Stoichiometric analysis of oligomeric states of three class-A GPCRs, chemokine-CXCR4, dopamine-D2, and prostaglandin-EP1 receptors, on living cells.

G-protein-coupled receptors (GPCRs) form the largest family of transmembrane receptors, and their oligomerization has been suggested to be related to their functions. Despite extensive studies, their oligomeric states are highly controversial. One of the reasons is the overestimation of oligomerization by conventional methods. We recently established a stoichiometric analysis method for precisely determining the oligomeric state of membrane proteins on living cells with the combined use of the coiled-coil labeling method and a spectral imaging technique and showed that the prototypical class-A GPCR β2 -adrenergic receptor (β2 AR) did not form functional oligomers. In this study, we expanded our study to three well-studied class-A GPCRs: C-X-C chemokine receptor of stromal cell-derived factor-1α (CXCR4), dopamine receptor D2 short isotype (D2R), and prostaglandin E receptor subtype 1 (EP1R). We found that these receptors did not form constitutive homooligomers. The receptors exhibited calcium signaling upon agonist stimulation as monomers, although CXCR4 and EP1R gradually clustered after fast signaling. We conclude that homooligomerization is not necessary for the signal transductions of these four class-A GPCRs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.