ABSTRACT
Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.
Subject(s)
Antineoplastic Agents , Pancreatic Neoplasms , Purines , Smoothened Receptor , Humans , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/metabolism , Purines/chemistry , Purines/pharmacology , Purines/chemical synthesis , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Ligands , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Animals , Mice , Structure-Activity Relationship , Drug Screening Assays, Antitumor , Molecular Structure , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , HEK293 Cells , Cell Line, Tumor , NIH 3T3 Cells , Molecular Docking Simulation , Hedgehog Proteins/metabolism , Hedgehog Proteins/antagonists & inhibitorsABSTRACT
Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.
ABSTRACT
The Frizzled family of transmembrane receptors (FZD1-10) belongs to the class F of G protein-coupled receptors (GPCRs). FZDs bind to and are activated by Wingless/Int1 (WNT) proteins. The WNT/FZD signaling system regulates crucial aspects of developmental biology and stem-cell regulation. Dysregulation of WNT/FZD communication can lead to developmental defects and diseases such as cancer and fibrosis. Recent insight into the activation mechanisms of FZDs has underlined that protein dynamics and conserved microswitches are essential for FZD-mediated information flow and build the basis for targeting these receptors pharmacologically. In this review, we summarize recent advances in our understanding of FZD activation, and how novel concepts merge and collide with existing dogmas in the field.
Subject(s)
Frizzled Receptors , Humans , Frizzled Receptors/metabolism , Animals , Wnt Signaling Pathway/drug effects , Wnt Proteins/metabolismABSTRACT
The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
Subject(s)
Bacterial Toxins , Boron Compounds , Clostridioides difficile , Clostridium Infections , Humans , Cryoelectron MicroscopyABSTRACT
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive. Here we employ a structure-driven mutagenesis approach in combination with an extensive panel of functional signaling readouts to investigate the importance of conserved state-stabilizing residues in FZD5 for signal specification. Similar data were obtained for FZD4 and FZD10 suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD5 mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD5 and FZDs in general prefer coupling to DVL rather than heterotrimeric G proteins and that distinct active state micro-switches in the receptor are essential for pathway selection arguing for conformational changes in the receptor protein defining transducer selectivity.
Subject(s)
Molecular Dynamics Simulation , Signal Transduction , Humans , Molecular Conformation , Mutagenesis , TransducersABSTRACT
Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD5-DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5-DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5-DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5. The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.
Subject(s)
Frizzled Receptors , Signal Transduction , Frizzled Receptors/metabolism , Ligands , Receptors, G-Protein-Coupled/metabolism , Wnt Signaling Pathway , Dishevelled Proteins/metabolism , Phosphoproteins/metabolismABSTRACT
BACKGROUND AND PURPOSE: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt-FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7 , and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. EXPERIMENTAL APPROACH: SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7 , preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. KEY RESULTS: With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd . Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. CONCLUSIONS AND IMPLICATIONS: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt-FZD7 binding should be performed using receptors expressed under endogenous promotion.
ABSTRACT
Smoothened (SMO) is a G protein-coupled receptor (GPCR) that mediates Hedgehog (Hh) signaling. SMO activity is regulated following the binding of Hh to the transmembrane protein Patched. Overactive SMO signaling is oncogenic, and hence this receptor is a target for several marketed drugs. However, development of new SMO ligands has been hampered by the fact that current radioligand and fluorescence-based binding assays are not high-throughput scalable. Here, we demonstrate two Nanoluciferase (Nluc) bioluminescence resonance energy transfer-based ligand binding assays (NanoBRET and NanoBiT/BRET) which provide a sensitive and high-throughput-compatible tool in drug screening efforts. In the described assays, SMO is N-terminally tagged either with full-length nanoluciferase or the partial HiBiT sequence, and subsequently binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between the receptor and the fluorescent ligand. Additionally, the assay allows performing competition binding experiments using commercially available SMO ligands, such as the SMO agonist SAG1.3.
Subject(s)
Smoothened Receptor/metabolism , Biological Assay , Hedgehog Proteins/genetics , Ligands , Receptors, G-Protein-Coupled/genetics , Signal Transduction , Smoothened Receptor/geneticsABSTRACT
The Frizzled (FZD) family of WNT receptors consists of ten paralogues in mammals. They belong to the superfamily of G protein-coupled receptors and regulate crucial processes during embryonic development. Dysregulated FZD signaling leads to disease, most prominently to diverse forms of cancer, which renders these receptors attractive for drug discovery. Recent advances in assay development and the design of genetically encoded biosensors monitoring ligand-receptor interaction, conformational dynamics, and protein-protein interaction have allowed for a better pharmacological understanding of WNT/FZD signal transduction and open novel avenues for mechanism-based drug discovery and screening. In this chapter, we summarize the recent progress in the molecular dissection of FZD activation based on advanced biosensors.
Subject(s)
Frizzled Receptors , Wnt Proteins , Animals , Cell Membrane , Frizzled Receptors/genetics , Humans , Ligands , Wnt Proteins/genetics , Wnt Signaling PathwayABSTRACT
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.
Subject(s)
Frizzled Receptors/chemistry , Frizzled Receptors/metabolism , Smoothened Receptor/chemistry , Binding Sites , Bioluminescence Resonance Energy Transfer Techniques , Boron Compounds/chemistry , Cryoelectron Microscopy , Cyclic AMP-Dependent Protein Kinases/metabolism , Frizzled Receptors/genetics , Humans , Molecular Dynamics Simulation , Mutation , Phosphoproteins/metabolism , Protein Conformation , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Veratrum Alkaloids/chemistry , Veratrum Alkaloids/metabolismABSTRACT
The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors-the ten mammalian Frizzleds (FZDs)-remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.
ABSTRACT
Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions.
Subject(s)
Fragile X Mental Retardation Protein , Fragile X Syndrome , Animals , Mice , Mice, Knockout , Mitochondrial Proteins/genetics , Proteomics , SynapsesABSTRACT
The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1-10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/ß-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.
Subject(s)
Frizzled Receptors/metabolism , Wnt Signaling Pathway , Wnt3A Protein/metabolism , Animals , Frizzled Receptors/analysis , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Mice , Microscopy, Confocal/methods , Protein Interaction Maps , Protein Transport , Wnt3A Protein/analysis , XenopusABSTRACT
WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.
Subject(s)
Dishevelled Proteins/metabolism , Drug Discovery/methods , Frizzled Receptors/agonists , Protein Interaction Domains and Motifs/drug effects , Pyridines/chemistry , Thiophenes/chemistry , Wnt Signaling Pathway/drug effects , Cell Membrane/metabolism , Frizzled Receptors/chemistry , Frizzled Receptors/metabolism , HEK293 Cells , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Targeted Therapy/methods , Morpholines/pharmacology , Proof of Concept Study , Purines/pharmacology , Pyridines/pharmacology , Smoothened Receptor/agonists , Structure-Activity Relationship , Thiophenes/pharmacologyABSTRACT
Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.
Subject(s)
Binding Sites/genetics , Biological Assay/methods , Signal Transduction/drug effects , Smoothened Receptor/antagonists & inhibitors , Veratrum Alkaloids/pharmacology , Bioluminescence Resonance Energy Transfer Techniques/methods , Boron Compounds/chemistry , Cinnamates/pharmacology , Drug Discovery/methods , Gene Knockout Techniques , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Ligands , Luciferases/chemistry , Morpholines/pharmacology , Nanostructures/chemistry , Purines/pharmacology , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Veratrum Alkaloids/chemistryABSTRACT
A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, 1H-15N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30 µg/mL) was comparable with staurosporine (10 µM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0-72 h); toxicity was not evident at 30 µg/mL.
Subject(s)
Apocynaceae/chemistry , Cytotoxins/pharmacology , Embryo, Nonmammalian/pathology , Indole Alkaloids/pharmacology , Plant Bark/chemistry , Plant Extracts/pharmacology , Zebrafish/growth & development , Animals , Embryo, Nonmammalian/drug effects , Humans , THP-1 CellsABSTRACT
Class F receptors, including the ten Frizzleds (FZD1-10 ) and SMO, mediate the effects of WNTs and hedgehog proteins and belong to the superfamily of G protein-coupled receptors (GPCRs). While the recent, high-resolution insight into mechanisms of GPCR activation provides a better understanding of receptor activation in Class A, B and C GPCRs, it remains unclear how Class F receptors bind their ligands, how ligand binding is translated to receptor activation and how signal initiation and specification are achieved. Here, we summarize recent efforts in elucidating Class F receptor structure and activation mechanisms and critically discuss the progress made in this area. A better understanding of the activation mechanisms of Class F receptors is required to engage in mechanism-based and structure-guided drug discovery to exploit the large therapeutic potential of targeting these receptors pharmacologically.
Subject(s)
Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Allosteric Regulation , Animals , Humans , Ligands , Receptors, G-Protein-Coupled/classification , Signal TransductionABSTRACT
The class Frizzled (FZD) or class F of G protein-coupled receptors consists of 10 FZD paralogues and Smoothened (SMO). FZDs coordinate wingless/Int-1 signaling and SMO mediates Hedgehog signaling. Class F receptor signaling is intrinsically important for embryonic development and its dysregulation leads to diseases, including diverse forms of tumors. With regard to the importance of class F signaling in human disease, these receptors provide an attractive target for therapeutics, exemplified by the use of SMO antagonists for the treatment of basal cell carcinoma. Here, we review recent structural insights in combination with a more detailed functional understanding of class F receptor activation, G protein coupling, conformation-based functional selectivity, and mechanistic details of activating cancer mutations, which will lay the basis for further development of class F-targeting small molecules for human therapy. SIGNIFICANCE STATEMENT: Stimulated by recent insights into the activation mechanisms of class F receptors from structural and functional analysis of Frizzled and Smoothened, we aim to summarize what we know about the molecular details of ligand binding, agonist-driven conformational changes, and class F receptor activation. A better understanding of receptor activation mechanisms will allow us to engage in structure- and mechanism-driven drug discovery with the potential to develop more isoform-selective and potentially pathway-selective drugs for human therapy.
Subject(s)
Embryonic Development/drug effects , Frizzled Receptors/agonists , Ligands , Molecular Targeted Therapy/methods , Smoothened Receptor/agonists , Animals , Drug Discovery/methods , Embryonic Development/physiology , Frizzled Receptors/metabolism , Hedgehog Proteins/metabolism , Humans , Protein Binding , Signal Transduction/drug effects , Signal Transduction/physiology , Smoothened Receptor/metabolism , Structure-Activity Relationship , Wnt Proteins/metabolismABSTRACT
GPR61 is an orphan receptor that belongs to Class A of G-protein-coupled receptors. It has been reported that GPR61 has a constitutive activity and couples to Gαs. In the present study, we characterized GPR61 function and ligand binding by experimental and molecular docking studies. We demonstrated that heterologous expression of GPR61 in HEK293 cells enhanced the cAMP synthesis response to forskolin, whereas the basal cAMP synthesis was unaffected. 5-(Nonyloxy)tryptamine inhibited forskolin-stimulated cAMP production in GPR61-expressing HEK293 cells. These studies highlight that the intrinsic activity of this receptor is only measurable following its synergy with Gαs.