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.     

Molecular Analysis of the Prostacyclin Receptor’s Interaction with the PDZ1 Domain of Its Adaptor Protein PDZK1

The prostanoid prostacyclin, or prostaglandin I₂, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK⁴¹¹IAACSLC⁴¹⁷) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (⁴¹¹IAACSLC⁴¹⁷). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Molecular Analysis of the Functional Role of β-Adrenergic Receptor Kinase 1 Amino-Terminal

Abstract

Receptor phosphorylation is a key step in the process of rapid desensitization. β-adrenergic receptor kinase (βARK) is a specific receptor kinase that is known to phosphorylate and induce desensitization of several G-coupled receptors only when they are occupied by their agonists. In the present study we have done several modifications to the amino-terminal of βARK1, in order to clarify its functional role. The recombinant mutants were tested for their ability to phosphorylate rhodopsin present in purified bovine ROS membranes which serves as a substrate for βARK1. Their expression levels were detected by Western blot analysis. We found that when the amino-terminal of βARK1 is modified its expression level is very low, hence it is not able to phosphorylate over the basal. These findings suggest that this region is crucial for the normal processing of the protein.     

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.     

Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.     

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a β-Sheet Bundle in Lipid Bilayers

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8–65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. ¹³C and ¹⁵N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell’s Response to LPA

Lysophosphatidic acid (LPA) mediates diverse cellular responses through the activation of at least six LPA receptors – LPA_1–6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA₁ contains a PDZ binding motif (SVV) identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA₁ but not that of other LPA receptors. LPA₁ colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA₁ to EEA1 early endosomes and promoted LPA₁ mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA₁ and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.     

Z-band Alternatively Spliced PDZ Motif Protein (ZASP) Is the Major O-Linked β-N-Acetylglucosamine-substituted Protein in Human Heart Myofibrils

We studied O-linked β-N-acetylglucosamine (O-GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O-GlcNAc with UDP-N-azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O-GlcNAc. All three methods showed that O-GlcNAc modification was predominantly in a group of bands ∼90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O-GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O-GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O-GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O-GlcNAcylated proteins in mouse heart myofibrils.     

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070–12082). Here, we identify the mediators of CB1 receptor internalization and {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced signaling mechanism for CB1 wherein β-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-β-arrestin 1 upon {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569. These results demonstrate for the first time the involvement of β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.     

Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin

Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α_2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α₂ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α_2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α_2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α_2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α_2AAR endocytosis and that α_2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α_2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α_2AARs. Importantly, we show that α₂AR agonist-mediated α_2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α₂ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α_2AARs and accelerating α_2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α_2AARs.     

GRIN1 Regulates ��-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein α-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence ²⁶⁷GSKEK²⁷¹ within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Gα interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Gα. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Functional Receptor Coupling to Gi Is A Mechanism of Agonist-Promoted Desensitization of the β2-Adrenergic Receptor

Abstract

The β₂-adrenergic receptor (β₂AR) couples to G_s, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β₂AR also couple to G_i after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β₂AR-G_i coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β₂AR or β₂AR and G_iα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β₂AR desensitization. Membrane AC activities showed that G_iα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing G_iα2. in the absence of such overexpression, β₂AR desensitization was 23 ± 7%, while with 5-fold G_iα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of G_i on desensitization was receptor-specific, in that forskolin responses were not altered by G_iα2 overexpression. Thus, acquired β₂AR coupling to G_i is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G_i levels contribute to β₂AR dysfunction.     

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background

Agonistic autoantibodies directed at the α₁-adrenergic receptor (α₁-AAB) have been described in patients with hypertension. We implied earlier that α₁-AAB might have a mechanistic role and could represent a therapeutic target.

Methodology/Principal Findings

To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α₁-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α₁-adrenergic receptor antibodies (α₁-AB). Patient α₁-AAB and rabbit α₁-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α_1A-adrenergic receptor were incubated with patient α₁-AAB and rabbit α₁-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α₁-AAB and rabbit α₁-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca²⁺ in cardiomyocytes and induce mesentery artery segment contraction.

Conclusions/Significance

Patient α₁-AAB and rabbit α₁-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α₁-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.     

Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

The β₁-adrenergic receptor (β₁AR) is the predominant βAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human β₁AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293_i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn¹⁵. Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg³¹ and Leu³² and Pro⁵² and Leu⁵³, which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the βAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg³¹–Leu³² cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface β₁ARs.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.