Signaling diversity of mu- and delta- opioid receptor ligands: Re-evaluating the benefits of β-arrestin/G protein signaling bias

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids. Advances in the field have led to the development of biased agonists based on hypotheses that allocated desired and undesired effects to specific signaling pathways. However, the prevalent hypothesis associating β-arrestin to opioid side effects was recently challenged and multiple of the newly developed biased drugs may not display the superior side effects profile that was sought. Moreover, biased agonism at opioid receptors is now known to be time- and cell-dependent, which adds a new layer of complexity for bias estimation. Here, we first review the signaling mechanisms underlying desired and undesired effects of opioids. We then describe biased agonism at opioid receptors and discuss the different perspectives that support the desired and undesired effects of opioids in view of exploiting biased signaling for therapeutic purposes. Finally, we explore how signaling kinetics and cellular background can influence the magnitude and directionality of bias at those receptors.     

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β₁-adrenergic (β₁AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β₁AR. Immunization with the β₁AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β₁AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β₁AR to which the mAbs bind. The anti-β₁AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β₁AR agonism.     

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β₁-adrenergic (β₁AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β₁AR. Immunization with the β₁AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β₁AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β₁AR to which the mAbs bind. The anti-β₁AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β₁AR agonism.     

Homo- and hetero-oligomerization of β2-adrenergic receptor in receptor trafficking,signaling pathways and receptor pharmacology

The β₂-adrenergic receptor (β₂AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β₂AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β₂AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β₂AR oligomerization with focus on the implications of the receptor–receptor interactions to β₂AR trafficking, pharmacology and downstream signal transduction pathways.     

A receptor for meningococcus: eliciting β-arrestin signaling for barrier breaching

In a recent issue of Cell, Coureuil et?al. (2010) show that type IV pili of Neisseria meningitidis-the causative bacterium of cerebrospinal meningitis-bind to the endothelial β2-adrenoceptor and elicit biased β-arrestin signaling resulting in microvilli formation and paracytotic bacterial migration.     

Ability of CK2β to selectively regulate cellular protein kinases

The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it has been reported that although Wee1 lacks the conserved binding motif recognised by beta-TrCP, the CDK-catalysed phosphorylation of Wee1 at Ser123 creates a phosphodegron and primes phosphorylation of two other protein kinases, polo-like kinase 1 (PLK1) and protein kinase CK2, which create two additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta-subunit of protein kinase CK2 can interact with Wee1 in high molecular mass complexes. Indirect immunofluorescence microscopy revealled subcellular co-localisation of CK2beta and Wee1 in the nucleus. Moreover, in vitro phosphorylation assays showed that CK2beta indirectly up-regulates the activity of CDK1 with respect to histone H1 phosphorylation by inhibiting Wee1 kinase. These findings support the view that CK2beta regulates various intracellular processes by modulating the activity of protein kinases that are distinct from CK2 and that protein kinase CK2 plays an important role in events related to the regulation of cell cycle progression as a tetrameric enzyme but also through the individual subunits.     

Serotonin receptor signaling and regulation via β-arrestins

Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have "functional selectivity". The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.     

Serotonin receptor signaling and regulation via β-arrestins

Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have “functional selectivity”. The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.     

Analysis of L-DOPA and droxidopa binding to human β2-adrenergic receptor

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β₂-adrenergic receptor (β₂AR). The 3.2 Å x-ray crystal structure of human β₂AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β₂AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β₂AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β₂AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β₂AR-ligand complexes performed here.     

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization,efficient signaling,and interaction with β-arrestin 2

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and β-arrestin (β-arr 319–418) and by means of cell lines that expressed various endogenous levels of β-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and β-arrestin and even more profoundly in cells lacking both β-arrestin1 and β-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with β-arrestin2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the Slovenian Ministry of Higher Education, Science, and Technology (grant number 0406-007) and a Slovenian-Danish collaboration grant (BI-DK/06-07-007).     

Rapid dephosphorylation of G protein-coupled receptors by protein phosphatase 1β is required for termination of β-arrestin-dependent signaling

Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that β-arrestin redistributes to phosphorylated GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. β-Arrestin in turn serves as a scaffold to initiate a second wave of signaling. Here, we report a molecular mechanism that regulates the termination of unconventional β-arrestin-dependent GPCR signaling. We identify protein phosphatase 1β (PP1β) as a phosphatase for the cluster of phosphorylated threonines ((353)TTETQRT(359)) within the sst(2A) somatostatin receptor carboxyl terminus that mediates β-arrestin binding using siRNA knock-down screening. We show that PP1β-mediated sst(2A) dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1β activity, we find that somatostatin- and substance P-induced but not epidermal growth factor-induced ERK activation was aberrantly enhanced and prolonged. Thus, we demonstrate a novel mechanism for fine tuning unconventional β-arrestin-dependent GPCR signaling in that recruitment of PP1β to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the β-arrestin-GPCR complex.     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Specific interactions between Epac1, β-arrestin2 and PDE4D5 regulate β-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling

β1 and β2 adrenergic receptors (βARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both βAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of βARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein β-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that β-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of β-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon β1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with β-arrestin2 following β2AR activation. Dissociation of the PDE4D5–β-arrestin2 complex allowed the recruitment of Epac1 to β2AR and induced a switch from β2AR non-hypertrophic signaling to a β1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which βAR subtypes exert opposing effects.     

Prokineticin receptors interact unselectively with several G protein subtypes but bind selectively to β-arrestin 2

Prokineticin 1 (pk1) and prokineticin 2 (pk2) interact with two structurally related G-protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). Cellular signalling studies show that the activated receptors can evoke Ca²⁺-mobilization, pertussis toxin-sensitive ERK phosphorylation, and intracellular cAMP accumulation, which suggests the partecipation of several G protein subtypes, such as G_q/11, G_i/o and G_s. However, direct interactions with these transduction proteins have not been studied yet. Here we measured by bioluminescence resonance energy transfer (BRET) the association of PKR1 and PKR2 with different heterotrimeric Gα proteins in response to pk1 and pk2 activation. Using host-cell lines carrying gene deletions of Gα_q/11 or Gα_s, and pertussis toxin treatment to abolish the receptor interactions with Gα_i/o, we determined that both receptors could couple with comparable efficiency to G_q/11 and G_i/o, but far less efficiently to G_s or other pertussis toxin-insensitive G proteins. We also used BRET methodology to assess the association of prokineticin receptors with β-arrestin isoforms. Fluorescent versions of the isoforms were transfected both in HEK293 cells and in double KO β-arrestin 1/2 mouse fibroblasts, to study receptor interaction with the reconstituted individual β-arrestins without background expression of the endogenous genes. Both receptors formed stable BRET-emitting complexes with β-arrestin 2 but not with β-arrestin 1, indicating strong selectivity for the former. In all the studied transducer interactions and in both receptors, pk2 was more potent than pk1 in promoting receptor binding to transduction proteins.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.