Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D1 receptors

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/β, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D₁ receptor. Here, we report on the cloning of two further dopamine D₁ receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D₁-like receptors but shows some pharmacological similarities to invertebrate “DOP1” dopamine D₁-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate “INDR”-like dopamine D₁-like receptors.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α_1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α_1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α_1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α_1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α_1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).     

Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β₁-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β₁-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β₁-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.     

Receptor,Ligand and Transducer Contributions to Dopamine D2 Receptor Functional Selectivity

Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor’s signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D₂R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and β-arrestins. D₂R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D₂R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D₂R mutants in which G protein-dependent (^[Gprot]D₂R) and β-arrestin-dependent signaling (^[βarr]D₂R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D₂R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating ^[Gprot]D₂R and ^[βarr]D₂R conferred control of partial agonism at G protein and/or β-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and β-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D₂R functional selectivity and point to means by which novel in vivo therapies could be modeled.     

Non-equivalent Ligand Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A KEY DETERMINANT OF AGONIST EFFICACY*

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4β2)₂α4 and (α4β2)₂β2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4β2)₂α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4β2)₂α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)₂α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)₂α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4β2)₂α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Assembly of a Ca2+-dependent BK channel signaling complex by binding to beta2 adrenergic receptor

Large-conductance voltage and Ca²⁺-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β-adrenergic receptors and protein kinase A (PKA_c) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2-adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L-type Ca²⁺ channels (Ca_v1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca²⁺- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β-adrenergic regulation of membrane excitability.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

A novel enzyme-linked receptor assay (ELRA) based on β₂-adrenergic receptor (β₂-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β₂-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β₂-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β₂-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.     

Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: INSIGHTS FROM INTACT CELL PHOSPHORYLATION AND RECRUITMENT ASSAYS*

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α_2A-adrenergic receptors (α_2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α_2AAR with EC₅₀ values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu⁴, Val⁷, Leu⁸, Val¹¹, and Ser¹², directly interacts with receptors, whereas residues such as Asp¹⁰, Tyr¹³, Ala¹⁶, Met¹⁷, Gly⁴⁷⁵, Val⁴⁷⁷, and Ile⁴⁸⁵ are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.     

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β₂-adrenergic receptor (β₂AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β₂AR. We show that the select ligands bind preferentially to different predicted conformers of β₂AR, and identify a role of β₂AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as “computational probes” to systematically identify protein conformers with likely biological significance.     

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.     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα_i free of Gβγ providing an unusual scaffold for the “G-switch” and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα_i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα_i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α₂-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα_i1-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα_i-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα_i and/or Gα_o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

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.     

Cardiac Effects of Attenuating Gsα - Dependent Signaling

Aims

Inhibition of β-adrenergic signalling plays a key role in treatment of heart failure. Gsα is essential for β-adrenergic signal transduction. In order to reduce side-effects of beta-adrenergic inhibition diminishing β-adrenergic signalling in the heart at the level of Gsα is a promising option.

Methods and Results

We analyzed the influence of Gsα on regulation of myocardial function and development of cardiac hypertrophy, using a transgenic mouse model (C57BL6/J mice) overexpressing a dominant negative Gsα-mutant under control of the α-MHC-promotor. Cardiac phenotype was characterized in vivo and in vitro and under acute and chronic β-adrenergic stimulation. At rest, Gsα-DN-mice showed bradycardia (602 ± 13 vs. 660 ± 17 bpm, p<0.05) and decreased dp/dt_max (5037 ± 546- vs. 6835 ± 505 mmHg/s, p = 0.02). No significant differences were found regarding ejection fraction, heart weight and cardiomyocyte size. β-blockade by propranolol revealed no baseline differences of hemodynamic parameters between wildtype and Gsα-DN-mice. Acute adrenergic stimulation resulted in decreased β-adrenergic responsiveness in Gsα-DN-mice. Under chronic adrenergic stimulation, wildtype mice developed myocardial hypertrophy associated with increase of LV/BW-ratio by 23% (4.4 ± 0.2 vs. 3.5 ± 0.1 mg/g, p<0.01) and cardiac myocyte size by 24% (14927 ± 442 px vs. 12013 ± 583 px, p<0.001). In contrast, both parameters were unchanged in Gsα-DN-mice after chronic isoproterenol stimulation.

Conclusion

Overexpression of a dominant negative mutant of Gsα leads to decreased β-adrenergic responsiveness and is protective against isoproterenol-induced hypertrophy. Thus, Gsα-DN-mice provide novel insights into β-adrenergic signal transduction and its modulation in myocardial overload and failure.     

Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D₁-like family of dopamine receptors and inputs of aversion coming from neurons containing the D₂-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D₁ reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D₂ receptors function, we present evidence of σ₁ receptor molecular and functional interaction with dopamine D₂ receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D₂ receptors (the long isoform of the D₂ receptor) can complex with σ₁ receptors, a result that is specific to D₂ receptors, as D₃ and D₄ receptors did not form heteromers. We demonstrate that the σ₁-D₂ receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ₁ -D₂ receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ₁ knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D₂ receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D₁ and D₂ receptor containing neurons in the brain.