Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone.
ABSTRACT: G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward β-arrestin-1 and β-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting β-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.
Project description:Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and ?-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or ?-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that ?-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/?-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.
Project description:Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through ?-arrestins. As such, monitoring ?-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including ?-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-?-arrestin interaction via ?-lactamase enzyme fragment complementation. Inter alia, ?-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of ?-lactamase (? and ?) were fused to ?-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (?-arrestin-? and GPCR-?), and this inducible interaction was measured through reconstituted ?-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.
Project description:G protein-coupled receptors (GPCRs) respond to diversified extracellular stimuli to modulate cellular function. Despite extensive studies investigating the regulation of single GPCR signaling cascades, the effects of concomitant GPCR activation on downstream signaling and cellular function remain unclear.We aimed to characterize the cellular mechanism by which GPCR crosstalk regulates mitogen-activated protein kinase (MAPK) activation.Adrenergic receptors on cardiac fibroblasts were manipulated to examine the role of arrestin in the spatiotemporal regulation of extracellular signal-regulated kinase (ERK)1/2 MAPK signaling. We show a general mechanism in which arrestin activation by one GPCR is capable of regulating signaling originating from another GPCR. Activation of Gq coupled-receptor signaling leads to prolonged ERK1/2 MAPK phosphorylation, nuclear accumulation, and cellular proliferation. Interestingly, coactivation of these receptors with the beta-adrenergic receptors induced transient ERK signaling localized within the cytosol, which attenuated cell proliferation. Further studies revealed that recruitment of arrestin3 to the beta2-adrenergic receptor orchestrates the sequestration of Gq-coupled receptor-induced ERK to the cytosol through direct binding of ERK to arrestin.This is the first evidence showing that arrestin3 acts as a coordinator to integrate signals from multiple GPCRs. Our studies not only provide a novel mechanism explaining the integration of mitogenic signaling elicited by different GPCRs, but also underscore the critical role of signaling crosstalk among GPCRs in vivo.
Project description:?-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: ?-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-?-arrestin complexes. We show here, using ?1-adrenergic receptors, that ?-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and ?-arrestin-dependent MAP kinase signal. These results demonstrate ?-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a ?-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.
Project description:<h4>Aims</h4>Cardiac ?-adrenergic receptor (?AR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ?ARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists.<h4>Methods and results</h4>Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ?ARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled ?1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ?1AR and ?2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled ?2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ?2AR phosphorylation effectively inhibit ?AR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ?2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ?2AR in a ?-arrestin 2 dependent manner without promoting ?2AR endocytosis. The increased binding between ?2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ?2AR, inhibition of PDE4, or genetic ablation of PDE4D or ?-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ?-arrestin 2 or PDE4D gene also rescues ?-adrenergic stimuli-induced myocyte contractile function.<h4>Conclusions</h4>These data reveal essential roles of ?-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ?ARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.
Project description:The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11 and Gi/o to stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function CASR mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+i responses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSRR680G in HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca2+i responses. Moreover, this gain of function in MAPK activity occurred independently of Gq/11 and Gi/o and was mediated instead by a noncanonical pathway involving ?-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced ?-arrestin signaling by disrupting a salt bridge formed between Arg680 and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through ?-arrestin and the importance of the Arg680-Glu767 salt bridge in mediating signaling bias.
Project description:The neurohormone oxytocin is a key player in the modulation of reproductive and social behavioral traits, such as parental care. Recently, a correlation between different forms of oxytocin and behavioral phenotypes has been described in the New World Monkeys (NWMs). Here, we demonstrate that, compared with the Leu8OXT found in most placental mammals, the Cebidae Pro8OXT and Saguinus Val3Pro8OXT taxon-specific variants act as equi-efficacious agonists for the Gq-dependent pathway but are weaker agonists for the ?-arrestin engagement and subsequent endocytosis toward the oxytocin receptor (OXTR). Upon interaction with the AVPR1a, Pro8OXT and the common Leu8OXT yielded similar signaling profiles, being equally efficacious on Gq and ?-arrestin, while Val3Pro8OXT showed reduced relative efficacy toward ?-arrestin. Intranasal treatment with either of the variants increased maternal behavior and also promoted unusual paternal care in rats, as measured by pup-retrieval tests. We therefore suggest that Val3Pro8OXT and Pro8OXT are functional variants, which might have been evolutionarily co-opted as an essential part of the adaptive genetic repertoire that allowed the emergence of taxon-specific complex social behaviors, such as intense parental care in the Cebidae and the genus Saguinus.
Project description:The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP<sup>+</sup>) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP<sup>+</sup> glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP<sup>+</sup> glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP<sup>+</sup> glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP<sup>+</sup> glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female <i>Gfap</i>-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP<sup>+</sup> glia regulates cardiovascular functions in vivo.
Project description:?-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ?-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ?-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of ?-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes ?-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and ?-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ?-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ?-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-?-arrestin interaction, but also governs the structural rearrangements within ?-arrestins. Furthermore, we found that ?-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of ?-arrestins and reveal their novel role in receptor cross-talk.
Project description:?-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). ?-arrestin is widely believed to be activated by forming a stable and stoichiometric GPCR-?-arrestin scaffold complex, which requires and is driven by the phosphorylated tail of the GPCR. Here we demonstrate a distinct and additional mechanism of ?-arrestin activation that does not require stable GPCR-?-arrestin scaffolding or the GPCR tail. Instead, it occurs through transient engagement of the GPCR core, which destabilizes a conserved inter-domain charge network in ?-arrestin. This promotes capture of ?-arrestin at the plasma membrane and its accumulation in clathrin-coated endocytic structures (CCSs) after dissociation from the GPCR, requiring a series of interactions with membrane phosphoinositides and CCS-lattice proteins. ?-arrestin clustering in CCSs in the absence of the upstream activating GPCR is associated with a ?-arrestin-dependent component of the cellular ERK (extracellular signal-regulated kinase) response. These results delineate a discrete mechanism of cellular ?-arrestin function that is activated catalytically by GPCRs.