scholarly journals Re-evaluating how low intrinsic efficacy and apparent bias for G protein activation relates to the improved side effect profiles of new opioid agonists

2020 ◽  
Author(s):  
Edward L. Stahl ◽  
Laura M. Bohn
Keyword(s):  
G Protein ◽  
Therapeutic Window ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Protein Activation ◽  
Biased Agonism ◽  
Intrinsic Efficacy ◽  
Cellular Environment ◽  
G Protein Activation ◽  
G Protein Coupled

AbstractIn a recent report by Gillis et al., 2020 (1), it was suggested that low intrinsic agonism, and not biased agonism, leads to an improvement in the separation of potency in opioid-induced respiratory suppression versus antinociception. Although the compounds that were tested have been shown to display G protein signaling bias in prior publications, the authors conclude that since they cannot detect biased agonism in their cellular signaling studies the compounds are therefore not biased agonists. Rather, they conclude that it is low intrinsic efficacy that leads to the therapeutic window improvement. Intrinsic efficacy is the extent to which an agonist can stimulate a G protein-coupled receptor (GPCR) response in a system. The designation of full agonist is made to compounds that produce the highest observable activation in a system (maximum intrinsic efficacy); agonists producing some fraction of that response are considered partial agonists. The maximum response window is determined by the cellular environment, receptor and effector expression levels, and the amplification readout of the system. Biased agonism takes into consideration not only intrinsic efficacy, but also potency (concentration required to reach half maximal efficacy) of an agonist in an assay. Herein, the data published in the aforementioned manuscript was used to rederive the intrinsic efficacy and bias factors as ΔΔlog(τ/KA) and ΔΔlog(Emax/EC50). Based on this reanalysis, the data does not support the conclusion that biased agonism, favoring G protein signaling, was not present. Further, these observations agree with prior studies wherein oliceridine, PZM21 and SR-17018 were first described as biased agonists with improvement in antinociception over respiratory suppression in mice. Therefore, introducing G protein signaling bias may be a means to improve opioid analgesia while avoiding certain undesirable side effects.

scholarly journals Interferonβ-1b Induces the Expression of RGS1 a Negative Regulator of G-Protein Signaling

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Tiffany Tran ◽  
Pedro Paz ◽  
Sharlene Velichko ◽  
Jill Cifrese ◽  
Praveen Belur ◽  
...  
Keyword(s):  
G Protein ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Negative Regulator ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Peripheral Blood Mononuclear ◽  
Protein Activation ◽  
G Protein Activation ◽  
Multiple Sclerosis Patients

We present evidence of a link between interferonβ-1b (IFN-β) and G-protein signaling by demonstrating that IFN-β can induce the expression of the negative regulator of G-protein signaling 1 (RGS1). RGS1 reduces G-protein activation and immune cell migration by interacting with heterotrimeric G-proteins and enhancing their intrinsic GTPase activity. In this study, IFN-β treatment resulted in the induction of RGS1 in peripheral blood mononuclear cells (PBMCs), monocytes, T cells, and B cells. Induction of RGS1 by IFN-β was concentration dependent and observed at both the RNA and protein level. Other members of the RGS family were not induced by IFN-β, and induction of RGS1 required the activation of the IFN receptor. In addition, RGS1 induction was observed in PBMCs obtained from IFN-β-treated multiple sclerosis patients suggesting a possible, as yet unexplored, involvement of G-protein regulation in disease treatment. The upregulation of RGS1 by IFN-β has not been previously reported.

scholarly journals Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein

2017 ◽  
Vol 114 (48) ◽  
pp. E10319-E10328 ◽  
Author(s):  
Anthony Leyme ◽  
Arthur Marivin ◽  
Marcin Maziarz ◽  
Vincent DiGiacomo ◽  
Maria P. Papakonstantinou ◽  
...  
Keyword(s):  
G Protein ◽  
Rational Design ◽  
G Protein Signaling ◽  
Guanine Nucleotide ◽  
Protein Signaling ◽  
Nucleotide Exchange ◽  
Protein Activation ◽  
Conserved Sequence ◽  
G Protein Activation

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

scholarly journals Biased agonists of the chemokine receptor CXCR3 differentially drive formation of Gαi:β-arrestin complexes

2020 ◽  
Author(s):  
Kevin Zheng ◽  
Jeffrey S. Smith ◽  
Anmol Warman ◽  
Issac Choi ◽  
Jaimee N. Gundry ◽  
...  
Keyword(s):  
Complex Formation ◽  
Signaling Pathway ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Gpcr Signaling ◽  
Surface Receptors ◽  
Biased Agonism ◽  
G Protein Coupled

AbstractG-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, signal through the proximal effectors G proteins and β-arrestins to influence nearly every biological process. Classically, the G protein and β-arrestin signaling pathways have largely been considered separable. Recently, direct interactions between Gα protein and β-arrestin have been described and suggest a distinct GPCR signaling pathway. Within these newly described Gα:β-arrestin complexes, Gαi/o, but not other Gα protein subtypes, have been appreciated to directly interact with β-arrestin, regardless of canonical GPCR Gα protein subtype coupling. However it is unclear how biased agonists differentially regulate this newly described Gαi:β-arrestin interaction, if at all. Here we report that endogenous ligands (chemokines) of the GPCR CXCR3, CXCL9, CXCL10, and CXCL11, along with two small molecule biased CXCR3 agonists, differentially promote the formation of Gαi:β-arrestin complexes. The ability of CXCR3 agonists to form Gαi:β-arrestin complexes does not correlate well with either G protein signaling or β-arrestin recruitment. Conformational biosensors demonstrate that ligands that promoted Gαi:β-arrestin complex formation generated similar β-arrestin conformations. We find these Gαi:β-arrestin complexes can associate with CXCR3, but not with ERK. These findings further support that Gαi:β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of biased agonism.

scholarly journals Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

2011 ◽  
Vol 286 (22) ◽  
pp. 19259-19269 ◽  
Author(s):  
Il-Ha Lee ◽  
Sung-Hee Song ◽  
Craig R. Campbell ◽  
Sharad Kumar ◽  
David I. Cook ◽  
...  
Keyword(s):  
G Protein ◽  
Kinase Activity ◽  
Receptor Activation ◽  
Na Channel ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Α Subunits

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na+ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 (D110AGRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.

scholarly journals Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes

2021 ◽  
Vol 14 (673) ◽  
pp. eaax3053
Author(s):  
Mieke Metzemaekers ◽  
Anneleen Mortier ◽  
Alessandro Vacchini ◽  
Daiane Boff ◽  
Karen Yu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Chemotactic Activity ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Agonist Activity ◽  
N Terminus ◽  
G Protein Coupled

The inflammatory human chemokine CXCL5 interacts with the G protein–coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1–78), truncated CXCL5 [CXCL5(9–78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and β-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

scholarly journals Regulators of G protein Signaling (RGS) proteins (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

2020 ◽  
Vol 2020 (4) ◽  
Author(s):  
Katelin E. Ahlers-Dannen ◽  
Mohammed Alqinyah ◽  
Christopher Bodle ◽  
Josephine Bou Dagher ◽  
Bandana Chakravarti ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Signaling Cascades ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Rgs Domain ◽  
G Protein Coupled

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [160, 377, 411, 415, 416, 512, 519, 312, 6].

scholarly journals Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

2020 ◽  
Vol 295 (51) ◽  
pp. 17486-17496
Author(s):  
Christopher T. Schafer ◽  
Anthony Shumate ◽  
David L. Farrens
Keyword(s):  
G Protein ◽  
Protein Activation ◽  
Canonical Class ◽  
Equilibrium Binding ◽  
C Terminus ◽  
G Protein Activation ◽  
Ligand Discovery ◽  
Pharmaceutical Agents ◽  
G Protein Coupled ◽  
Covalently Bound

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

scholarly journals Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists

2020 ◽  
Vol 13 (625) ◽  
pp. eaaz3140 ◽  
Author(s):  
Alexander Gillis ◽  
Arisbel B. Gondin ◽  
Andrea Kliewer ◽  
Julie Sanchez ◽  
Herman D. Lim ◽  
...  
Keyword(s):  
G Protein ◽  
Side Effect ◽  
Opioid Analgesics ◽  
Receptor Activation ◽  
Alternative Mechanism ◽  
Protein Activation ◽  
Biased Agonism ◽  
Opioid Ligands ◽  
Intrinsic Efficacy ◽  
Respiratory Depressant

Biased agonism at G protein–coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus β-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as β-arrestin recruitment. At the μ-opioid receptor (MOR), G protein–biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in β-arrestin–mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and β-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

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