Agonist-directed desensitization of the β2-adrenergic receptor

The β(2)-adrenergic receptor (β(2)AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β(2)AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β(2)AR desensitization at the whole cell level.     

Multiple ligand-specific conformations of the β2-adrenergic receptor

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.     

Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry

Background

Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method

We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results

Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions

Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.     

Thyroid dependent maturation of β-adrenergic receptors in the rat lung

β-Adrenergic receptors were identified in membranes of fetal and postnatal rat lung with (?)-[³H]dihydroalprenolol, [³H]DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to adult levels by day 28 of postnatal life. The increase of β-adrenergic receptors occurring between postnatal days 15 and 28 was dependent on thyroxine (T4) in propylthiouracil treated pups. β-Adrenergic receptors on day 28 were identical in euthyroid (PTU + T4) as compared to normal control pups (489±31 and 491±30 femtomoles·mg^?1) however receptors were markedly reduced in 28 day hypothyroid pups (PTU alone), Bmax = 294±21.5, m±S.E. p<0.01. Treatment of the hypothyroid pups with T4 for three days on postnatal day 25 increased β-adrenergic receptors approximately two-fold by day 28. This thyroid hormone dependent increase in lung β-adrenergic receptors occurs between postnatal days 15 and 28 coincident with the known increase in thyroid gland activity in the rat pup.     

Molecular dynamics simulations of the effect of the G-protein and diffusible ligands on the β2-adrenergic receptor

G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β₂-adrenergic receptor (β₂AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist-β₂ adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β₂AR and the C-terminal end of the Gα_s subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β₂AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β₂AR and GαCT form a stable interaction that is destabilized by carazolol.     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.     

Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the β2-adrenergic receptor: Part 6

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human β₃-adrenergic receptor (AR) agonists. Result of structure–activity relationships (SAR) and cassette-dosing evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray structure of the β₂-adrenergic receptor.     

Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization

The oxytocin receptor (OTR) and the β₂-adrenergic receptor (β₂AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β₂AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β₂AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β₂AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β₂AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β₂AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β₂AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β₂AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β₂AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.     

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.     

Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor

Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β(2)-adrenergic receptor (β(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β(2)AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L(339)L(340) motif in the β(2)AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β(2)AR, which requires the interaction of RGGTA with the β(2)AR L(339)L(340) motif. Furthermore, the β(2)AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β(2)AR was dependent on the RGGTA-interacting L(339)L(340) motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β(2)AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.     

Modulation of myocardial stiffness by β-adrenergic stimulation--its role in normal and failing heart

The acute effects of beta-adrenergic stimulation on myocardial stiffness were evaluated. New-Zealand white rabbits were treated with saline (control group) or doxorubicin to induce heart failure (HF) (DOXO-HF group). Effects of isoprenaline (10(-10)-10(-5) M), a non-selective beta-adrenergic agonist, were tested in papillary muscles from both groups. In the control group, the effects of isoprenaline were also evaluated in the presence of a damaged endocardial endothelium, atenolol (beta(1)-adrenoceptor antagonist), ICI-118551 (beta(2)-adrenoceptor antagonist), KT-5720 (PKA inhibitor), L-NNA (NO-synthase inhibitor), or indomethacin (cyclooxygenase inhibitor). Passive length-tension relations were constructed before and after adding isoprenaline (10(-5) M). In the control group, isoprenaline increased resting muscle length up to 1.017+/-0.006 L/L(max). Correction of resting muscle length to its initial value resulted in a 28.5+/-3.1 % decrease of resting tension, indicating decreased muscle stiffness, as confirmed by the isoprenaline-induced right-downward shift of the passive length-tension relation. These effects were modulated by beta(1)- and beta(2)-adrenoceptors and PKA. In DOXO-HF group, the effect on myocardial stiffness was significantly decreased. We conclude that beta-adrenergic stimulation is a relevant mechanism of acute neurohumoral modulation of the diastolic function. Furthermore, this study clarifies the mechanisms by which myocardial stiffness is decreased.     

Exfoliation of the β-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed ‘exosomes’ into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, G_s, and the inhibitory, G_i, regulatory components) and β-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of G_s or G_i in the exosomes was detected by ADP ribosylation using [α-³²P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal G_s was almost as active as the membrane-derived G_s in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc⁻ cells, which lack a functional G_s. The ability of exosomal G_s to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane G_s. The density of β-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated β-adrenergic receptors seem to be impaired in their ability to couple to and activate G_s. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the β-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking

Very little is understood about the trafficking of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) to the plasma membrane. Rab guanosine triphosphatases (GTPases) are known to participate in the trafficking of various GPCRs via a direct interaction during the endocytic pathway, but whether this occurs in the anterograde pathway is unknown. We evaluated the potential interaction of Rab1, a GTPase known to regulate β2-adrenergic receptor (β2AR) trafficking, and its effect on export from the ER. Our results show that GTP-bound Rab1 interacts with the F(x)(6)LL motif of β2AR. Receptors lacking the interaction motif fail to traffic properly, suggesting that a direct interaction with Rab1 is required for β2AR anterograde trafficking.     

Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

Background

The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.

Methods

A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).

Results

Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.

Conclusion

Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.     

Role of the β<Subscript>3</Subscript>-adrenergic receptor subtype in catecholamine-induced myocardial remodeling

Alpha-synuclein (α-synuclein) aggregation and impairment of the Ubiquitin proteasome system (UPS) are implicated in Parkinson’s disease (PD) pathogenesis. While zinc (Zn) induces dopaminergic neurodegeneration resulting in PD phenotype, its effect on protein aggregation and UPS has not yet been deciphered. The current study investigated the role of α-synuclein aggregation and UPS in Zn-induced Parkinsonism. Additionally, levodopa (l-Dopa) response was assessed in Zn-induced Parkinsonian model to establish its closeness with idiopathic PD. Male Wistar rats were treated with zinc sulfate (Zn; 20 mg/kg; i.p.) twice weekly for 12 weeks along with respective controls. In few subsets, animals were subsequently treated with l-Dopa for 21 consecutive days following Zn exposure. A significant increase in total and free Zn content was observed in the substantia nigra of the brain of exposed groups. Zn treatment caused neurobehavioral anomalies, striatal dopamine decline, and dopaminergic neuronal cell loss accompanied with a marked increase in α-synuclein expression/aggregation and Ubiquitin-conjugated protein levels in the exposed groups. Zn exposure substantially reduced UPS-associated trypsin-like, chymotrypsin-like, and caspase-like activities along with the expression of SUG1 and β-5 subunits of UPS in the nigrostriatal tissues of exposed groups. l-Dopa treatment rescued from Zn-induced neurobehavioral deficits and restored dopamine levels towards normalcy; however, Zn-induced dopaminergic neuronal loss, reduction in tyrosine hydroxylase expression, and increase in oxidative stress were unaffected. The results suggest that Zn caused UPS impairment, resulting in α-synuclein aggregation subsequently leading to dopaminergic neurodegeneration, and that Zn-induced Parkinsonism exhibited positive l-Dopa response similar to sporadic PD.     

Influence of orthosteric ligand binding on the conformational dynamics of the β-2-adrenergic receptor via essential dynamics sampling simulation

The influence of the binding of orthosteric ligands on the conformational dynamics of the β-2-adrenoreceptor was identified using the molecular dynamics method. It was found that there was a small fraction of active states of the receptor in its apo (ligand free) ensemble. An analysis of the MD trajectories indicated that this spontaneous activation of the receptor was accompanied by the motion of its VI helix. Thus, the receptor’s constitutive activity is a direct result of its conformational dynamics. On the other hand, the binding of the full agonist resulted in a significant shift in the initial equilibrium towards its active state. Finally, the binding of the inverse agonist stabilized receptor in its inactive state. It is likely that the binding of the inverse agonists might be a universal method of the constitutive activity inhibition. Our results indicate that ligand binding redistributes preexisting conformational degrees of freedom (in accordance to the Monod-Wyman-Changeux Model), rather than causes an induced fit. Therefore, the ensemble of the biologically relevant receptors conformations has been encoded in its spatial structure and individual conformations from that ensemble and might be used by the cell according to the physiological behavior.