Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of α_2B- and β₂-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of α_2B-AR than β₂-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by α_2B-AR, but not β₂-AR, and arrested α_2B-AR in the TGN compartment. Co-immunoprecipitation revealed that both α_2B-AR and β₂-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked β₂-AR interaction with Rab8, whereas mutation of residues Val⁴³¹-Phe⁴³²-Asn⁴³³-Gln⁴³⁴, Pro⁴⁴⁷-Trp⁴⁴⁸, Gln⁴⁵⁰-Thr⁴⁵¹, and Trp⁴⁵³ in the C terminus impaired α_2B-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric β₂-AR carrying the α_2B-AR C terminus was similar to α_2B-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of α_2B-AR and β₂-AR and differentially modulates their traffic from the TGN to the cell surface.     

Rab26 Modulates the Cell Surface Transport of α2-Adrenergic Receptors from the Golgi

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α₂-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α_2A-AR and α_2B-AR, as well as ERK1/2 activation by α_2B-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α_2B-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α₂-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.     

Sorting of β1-Adrenergic Receptors Is Mediated by Pathways That Are Either Dependent on or Independent of Type I PDZ,Protein Kinase A (PKA), and SAP97

The β₁-adrenergic receptor (β₁-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β₁-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β₁-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085–5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β₁-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β₁-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β₁-AR. β₁-AR chimera containing the type I PDZ of the β₂-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β₁-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β₁-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β₁-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position −3 in the PDZ regulated whether the β₁-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β₁-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs.     

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β₁-adrenergic receptor (β₁-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β₁-AR. Recycling of the β₁-AR in AKAP5 knockdown cells was rescued by shRNA-resistant AKAP5. However, truncated mutants of AKAP5 with deletions in the domains involved in membrane targeting or in binding to calcineurin or PKA failed to restore the recycling of the β₁-AR, indicating that full-length AKAP5 was required. Furthermore, recycling of the β₁-AR in rat neonatal cardiac myocytes was dependent on targeting the AKAP5-PKA complex to the C-terminal tail of the β₁-AR. To analyze the role of AKAP5 more directly, recycling of the β₁-AR was determined in ventricular myocytes from AKAP5^−/− mice. In AKAP5^−/− myocytes, the agonist-internalized β₁-AR did not recycle, except when full-length AKAP5 was reintroduced. These data indicate that AKAP5 exerted specific and profound effects on β₁-AR recycling in mammalian cells. Biochemical or real time FRET-based imaging of cyclic AMP revealed that deletion of AKAP5 sensitized the cardiac β₁-AR signaling pathway to isoproterenol. Moreover, isoproterenol-mediated increase in contraction rate, surface area, or expression of β-myosin heavy chains was significantly greater in AKAP5^−/− myocytes than in AKAP5^+/+ myocytes. These results indicate a significant role for the AKAP5 scaffold in signaling and trafficking of the β₁-AR in cardiac myocytes and mammalian cells.     

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane

β₂-Adrenergic receptors (β₂-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β₂-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β₂-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β₂-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β₂-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β₂-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.     

A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell-Surface Expression of α2A-Adrenergic Receptor

Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65also modulates the cell-surface transport of α_2A-adrenergic receptor (α_2A-AR). Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α_2A-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of α_2A-AR, mutation of Lys65 to Ala and Arg produced opposing effects on α_2A-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α_2A-AR mutant K65A displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell-surface expression of α_2A-AR. These data also suggest that the ICL1 may possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms.     

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.     

Pivotal Role of the α2A-Adrenoceptor in Producing Inflammation and Organ Injury in a Rat Model of Sepsis

Background

Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-α production in Kupffer cells (KCs) through the activation of the α₂-AR. It is important to know which of the three α₂-AR subtypes (i.e., α_2A, α_2B or α_2C) is responsible for the upregulation of TNF-α production. The aim of this study was to determine the contribution of α_2A-AR in this process.

Methodology/Principal Findings

Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of α_2A-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific α_2A-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of α_2A-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the α_2A-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-α in cultured KCs, which was specifically inhibited by the α_2A-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-α gene expression in KCs and plasma TNF-α which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-α release via the α_2A-AR in vitro and in vivo. This potentiation of TNF-α release by NE was mediated through the α_2A-AR coupled Gαi protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-α, prevented multiple organ injury and significantly improved survival from 45% to 75%.

Conclusions/Significance

Our novel finding is that hyperresponsiveness to α₂-AR stimulation observed in sepsis is primarily due to an increase in α_2A-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α_2A-AR antagonist as a new therapy for sepsis.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.     

Neuronal protein trafficking associated with Alzheimer disease: From APP and BACE1 to glutamate receptors

Aberrant and/or cumulative amyloid-beta (Aβ) production, resulting from proteolytic processing of the amyloid precursor protein (APP) by β and γ-secretases, have been postulated to be a main etiological basis of Alzheimer disease (AD). A number of proteins influence the subcellular trafficking itinerary of APP and the β-site APP-cleaving enzyme (BACE1) between the cell surface, endosomes and the trans-Golgi network (TGN). Available evidence suggests that co-residence of APP and BACE1 in the endosomal compartments promotes amyloidogenesis. Retrograde transport of APP out of the endosome to the TGN reduces Aβ production, while APP routed to and kept at the cell surface enhances its non-amyloidogenic, α-secretase-mediated processing. Changes in post-Golgi membrane trafficking in aging neurons that may influence APP processing is particularly relevant to late-onset, idiopathic AD. Dystrophic axons are key features of AD pathology, and impaired axonal transport could play crucial roles in the pathogenesis of idiopathic AD. Recent evidence has also indicated that Aβ-induced synaptic defects and memory impairment could be explained by a loss of both AMPA and NMDA receptors through endocytosis. Detail understanding of factors that influence these neuronal trafficking processes will open up novel therapeutic avenues for preventing or delaying the onset of symptomatic AD.Key words: amyloid precursor protein (APP), β-site APP cleaving enzyme 1 (BACE1), endosome, glutamate receptors, trans-Golgi network (TGN)     

The β-blocker Nebivolol Is a GRK/β-arrestin Biased Agonist

Nebivolol, a third generation β-adrenoceptor (β-AR) antagonist (β-blocker), causes vasodilation by inducing nitric oxide (NO) production. The mechanism via which nebivolol induces NO production remains unknown, resulting in the genesis of much of the controversy regarding the pharmacological action of nebivolol. Carvedilol is another β-blocker that induces NO production. A prominent pharmacological mechanism of carvedilol is biased agonism that is independent of Gα_s and involves G protein-coupled receptor kinase (GRK)/β-arrestin signaling with downstream activation of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). Due to the pharmacological similarities between nebivolol and carvedilol, we hypothesized that nebivolol is also a GRK/β-arrestin biased agonist. We tested this hypothesis utilizing mouse embryonic fibroblasts (MEFs) that solely express β₂-ARs, and HL-1 cardiac myocytes that express β₁- and β₂-ARs and no detectable β₃-ARs. We confirmed previous reports that nebivolol does not significantly alter cAMP levels and thus is not a classical agonist. Moreover, in both cell types, nebivolol induced rapid internalization of β-ARs indicating that nebivolol is also not a classical β-blocker. Furthermore, nebivolol treatment resulted in a time-dependent phosphorylation of ERK that was indistinguishable from carvedilol and similar in duration, but not amplitude, to isoproterenol. Nebivolol-mediated phosphorylation of ERK was sensitive to propranolol (non-selective β-AR-blocker), AG1478 (EGFR inhibitor), indicating that the signaling emanates from β-ARs and involves the EGFR. Furthermore, in MEFs, nebivolol-mediated phosphorylation of ERK was sensitive to pharmacological inhibition of GRK2 as well as siRNA knockdown of β-arrestin 1/2. Additionally, nebivolol induced redistribution of β-arrestin 2 from a diffuse staining pattern into more intense punctate spots. We conclude that nebivolol is a β₂-AR, and likely β₁-AR, GRK/β-arrestin biased agonist, which suggests that some of the unique clinically beneficial effects of nebivolol may be due to biased agonism at β₁- and/or β₂-ARs.     

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET¹⁷⁸P), but not rat β-arrestin-1 (PER¹⁷⁷P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK¹⁷⁸P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species.     

Membrane Potential Controls the Efficacy of Catecholamine-induced β1-Adrenoceptor Activity

G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (V_M) in excitable tissues. These changes have been shown to alter receptor activation of certain G_i-and G_q-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the G_s-coupled β₁-adrenoreceptor (β₁-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by V_M. This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of β₂-AR activation, however, was weak compared with β₁-AR voltage-dependence. Drug efficacy is a major target of β₁-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of V_M and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied β₁-adrenoceptors on a very fast time scale.     

Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α₁-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α₁-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α₁-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca²⁺ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α₁-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α₁-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.     

In Silico Modeling of Human α2C-Adrenoreceptor Interaction with Filamin-2

Vascular smooth muscle α_2C-adrenoceptors (α_2C-ARs) mediate vasoconstriction of small blood vessels, especially arterioles. Studies of endogenous receptors in human arteriolar smooth muscle cells (referred to as microVSM) and transiently transfected receptors in heterologous HEK293 cells show that the α_2C-ARs are perinuclear receptors that translocate to the cell surface under cellular stress and elicit a biological response. Recent studies in microVSM unraveled a crucial role of Rap1A-Rho-ROCK-F-actin pathways in receptor translocation, and identified protein-protein interaction of α_2C-ARs with the actin binding protein filamin-2 as an essential step in the process. To better understand the molecular nature and specificity of this interaction, in this study, we constructed comparative models of human α_2C-AR and human filamin-2 proteins. Finally, we performed in silico protein-protein docking to provide a structural platform for the investigation of human α_2C-AR and filamin-2 interactions. We found that electrostatic interactions seem to play a key role in this complex formation which manifests in interactions between the C-terminal arginines of α_2C-ARs (particularly R454 and R456) and negatively charged residues from filamin-2 region between residues 1979 and 2206. Phylogenetic and sequence analysis showed that these interactions have evolved in warm-blooded animals.     

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β₂-adrenoceptor (β₂-AR) couples dually to G_s and G_i proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β₂-AR causes a switch in receptor coupling from G_s to G_i. More recent studies have demonstrated that phosphorylation of β₂-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the G_i coupling. We have previously shown that although most β₂-AR agonists cause both G_s and G_i activation, (R,R′)-fenoterol preferentially activates β₂-AR-G_s signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β₂-AR essential for G_s-biased signaling. Following stimulation with a β₂-AR-G_s-biased agonist (R,R′)-4′-aminofenoterol, the G_i disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β₂-AR. Interestingly, Y308F substitution on β₂-AR enabled (R,R′)-4′-aminofenoterol to activate G_i and to produce these responses in a pertussis toxin-sensitive manner without altering β₂-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β₂-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β₂-AR stabilize receptor conformations favoring the receptor-G_s protein coupling and subsequently result in G_s-biased agonism.     

γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABA_B receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABA_A receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABA_A receptors, where GHB acts as an agonist with an EC₅₀ of 140 nM. We investigated three neuronal cell types that express a tonic GABA_A receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABA_A receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABA_B receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.     

Autoantibodies against the β3-Adrenoceptor Protect from Cardiac Dysfunction in a Rat Model of Pressure Overload

β₃-adrenoceptors (β₃-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of β₁- and β₂-ARs. It has been previously demonstrated that autoantibodies against the β_1/β₂-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the β₃-AR (β₃-AR Abs) exist in the sera of patients with HF and to assess the effects of β₃-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of β₃-AR Abs in the sera of HF patients was screened by ELISA. β₃-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of β₃-AR Abs in the sera of HF patients were greatly increased, and β₃-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. β₃-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca²⁺ transient and membrane L-type Ca²⁺ current in cardiomyocytes. Our results demonstrated the existence of β₃-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.