Biotechnology of β-adrenergic receptors

The emergence of Biotechnology has provided pharmacologists with a variety of methods for investigating the structure, the function, and the regulation of membrane-bound receptors with a precision that was not imagined even five years ago. These new tools have been developed and used to analyze the known catecholamine β₁- and β₂ receptors and to discover and study a new subtype, the β-adrenergic receptor. We review here the salient features of each of these three receptors, compare their structural and functional properties, and propose models to explain their differential regulation in time and space. A whole family of proteins has now been found to share with the β-adrenergic receptors their most prominent features, including seven transmembrane domains and coupling with GTP-binding “G” proteins. We therefore propose that the biotechnology-based procedures developed for the β-adrenergic receptors will be well applicable to the other members of this “R₇G” family of receptors.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

β-Adrenergic receptors of frog erythrocytes

Following persistent stimulation of -adrenergic receptors of frog erythrocytes with (–)-isoproterenol, the cyclic adenosine 3,5-monophosphate-dependent protein kinase (cAMP-dependent protein kinase) (EC 2.7.1.37) was activated for several hours. This activation outlasted the duration of the increase of cAMP content. Following a persistant stimulation of -adrenergic receptors with isoproterenol, the phosphorylation of selective membrane proteins was increased. This increase in phosphorylation lasted longer than 4 hr but less than 12 hr. Between 2 and 4 hr after receptor stimulation the loss of -adrenergic receptor form plasma membrane was maximal, and the phosphorylation of two membrane proteins characterized by molecular weights of 60,000 and 38,000 daltons was selectively enhanced. In addition we found that isolated erythrocytes are capable of synthesizing RNA and polypeptides and that incubation with (–)-isoproterenol induces a longterm delayed increase of the synthesis of erythrocyte proteins. This increase in the synthesis of proteins appears to require new RNA synthesis. Thus the possibility can be entertained that this delayed increase in protein synthesis participates in the new synthesis of receptor and is operative in the termination of -adrenergic receptor subsensitivity elicited by a persistent stimulation with (–)-isoproterenol.     

Antinociceptive effects of novel epibatidine analogs through activation of α4β2 nicotinic receptors

The study of α4β2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4β2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4β2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury(CCI)-induced neuropathic pain in rats, which was sensitive to DHβE, a selective α4β2 subtype antagonist,indicating that its analgesic effect was mediated by the activation of the α4β2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4β2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.     

Internalization dissociates β2-adrenergic receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β(2)-adrenergic receptors (β(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β(2)ARs between subcellular compartments. BRET between β(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β(2)ARs and endosome markers increases. Energy transfer between β(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β(2)ARs associate transiently with each other in the plasma membrane, or that β(2)AR dimers or oligomers are actively disrupted during internalization.     

Characterization of interactions of 4-nitrophenylpropyl-N-alkylamine with ς receptors

Sigma receptors are small membrane proteins implicated in a number of pathophysiological conditions, including drug addiction, psychosis, and cancer; thus, small molecule inhibitors of sigma receptors have been proposed as potential pharmacotherapeutics for these diseases. We previously discovered that endogenous monochain N-alkyl sphingolipids, including d-erythro-sphingosine, sphinganine, and N,N-dimethylsphingosine, bind to the sigma-1 receptor at physiologically relevant concentrations [Ramachandran, S., et al. (2009) Eur. J. Pharmacol. 609, 19-26]. Here, we investigated several N-alkylamines of varying chain lengths as sigma receptor ligands. Although the K(I) values for N-alkylamines were found to be in the micromolar range, when N-3-phenylpropyl and N-3-(4-nitrophenyl)propyl derivatives of butylamine (1a and 1b, respectively), heptylamine (2a and 2b, respectively), dodecylamine (3a and 3b, respectively), and octadecylamine (4a and 4b, respectively) were evaluated as sigma receptor ligands, we found that these compounds exhibited nanomolar affinities with both sigma-1 and sigma-2 receptors. A screen of high-affinity ligands 2a, 2b, 3a, and 3b against a variety of other receptors and/or transporters confirmed these four compounds to be highly selective mixed sigma-1 and sigma-2 ligands. Additionally, in HEK-293 cells reconstituted with K(v)1.4 potassium channel and the sigma-1 receptor, these derivatives were able to inhibit the outward current from the channel, consistent with sigma receptor modulation. Finally, cytotoxicity assays showed that 2a, 2b, 3a, and 3b were highly potent against a number of cancer cell lines, demonstrating their potential utility as mixed sigma-1 and sigma-2 receptor anticancer agents.     

Shaping immune responses through the activation of dendritic cells–P2 receptors

Dendritic cells (DCs) activate and shape the adaptive immune response by capturing antigens, migrating to peripheral lymphoid organs where naïve T cells reside, expressing high levels of MHC and costimulatory molecules and secreting cytokines and chemokines. DCs are endowed with a high degree of functional plasticity and their functions are tightly regulated. Besides initiating adaptive immune responses, DCs play a key role in maintaining peripheral tolerance toward self-antigens. On the basis of the information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. A wide variety of signals from neighbouring cells affecting DC functional activity have been described. Here we will discuss the complex role of extracellular nucleotides in the regulation of DC function and the role of P2 receptors as possible tools to manipulate immune responses.     

Structure–activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y₁ while uridine containing dinucleotides have some level of agonist response on P2Y₂ and P2Y₆. The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.     

Is lipid signaling through cannabinoid 2 receptors part of a protective system?

The mammalian body has a highly developed immune system which guards against continuous invading protein attacks and aims at preventing, attenuating or repairing the inflicted damage. It is conceivable that through evolution analogous biological protective systems have been evolved against non-protein attacks. There is emerging evidence that lipid endocannabinoid signaling through cannabinoid 2 (CB₂) receptors may represent an example/part of such a protective system/armamentarium. Inflammation/tissue injury triggers rapid elevations in local endocannabinoid levels, which in turn regulate signaling responses in immune and other cells modulating their critical functions. Changes in endocannabinoid levels and/or CB₂ receptor expressions have been reported in almost all diseases affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, autoimmune, lung disorders to pain and cancer, and modulating CB₂ receptor activity holds tremendous therapeutic potential in these pathologies. While CB₂ receptor activation in general mediates immunosuppressive effects, which limit inflammation and associated tissue injury in large number of pathological conditions, in some disease states activation of the CB₂ receptor may enhance or even trigger tissue damage, which will also be discussed alongside the protective actions of the CB₂ receptor stimulation with endocannabinoids or synthetic agonists, and the possible biological mechanisms involved in these effects.     

Stimulation of β2-adrenergic receptors suppresses sterol synthesis in human mononuclear leukocytes

The β-adrenergic receptor mediating the inhibition of sterol synthesis by catecholamines in freshly isolated human mononuclear leukocytes was defined pharmacologically by using selective β₁- and β₂-agonists and -antagonists. Incubation of cells for 6 h in a medium containing lipid-depleted serum resulted in a 3-fold increase in the incorporation of [¹⁴C]acetate or tritiated water into sterols. The β-agonist (?)-isoproterenol was approximately equipotent with (?)-epinephrine and (?)-norepinephrine in suppressing sterol synthesis, yielding a sigmoidal log-dose-effect curve. Accordingly, the effects of the catecholamines were reversed by the β-antagonist (±)-propranolol. The β₂-agonists terbutaline and salbutamol inhibited sterol synthesis by 42 and 26%, respectively, at a concentration of 0.1 mmol/l. Contrary to that, the β₁-agonists prenalterol and dobutamine had no effect. In accordance with the influence of the agonists, the β₂-antagonist butoxamine, but not the β₁-antagonists atenolol, metoprolol and practolol, reversed the catecholamine action on sterol synthesis. The results provide evidence that catecholamines may regulate sterol synthesis by stimulating β₂-adrenergic receptors.     

Preparation of 123I-labeled 2′-iodospiperone and imaging of D2 dopamine receptors in the human brain using SPECT

[¹²³I]2′-ISP was readily prepared using a radioiodine exchange reaction with a radiochemical yield of approx. 50% after HPLC purification. The radiochemical purity of the product was more than 98% and the specific activity was 5.55–11.1 GBq/μmol. Biodistribution studies performed in mice indicated that injection of [¹²³I]2′-ISP with albumin produced a higher gastric uptake and a lower brain uptake than injection of the radioligand in a weakly acidic solution. In addition, toxicity tests performed in mice demonstrated that acute toxic effects would be very unlikely to be encountered if 2′-ISP was used for diagnostic purposes. A preliminary imaging study with [¹²³I]2′-ISP in a healthy human volunteer showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of D₂ dopamine receptors.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

How do G-protein-coupled receptors work? The case of metabotropic glutamate and GABA receptors

G-protein coupled receptors (GPCRs) represent the largest membrane proteins family in animal genomes. Being the receptors for most hormones and neurotransmitters, these proteins play a central role in intercellular communication. GPCRs can be classified into several groups based on the sequence similarity of their common structural feature: the heptahelical domain. The metabotropic receptors for the main neurotransmitters glutamate and gamma-aminobutyric acid (GABA) belong to the class III of GPCRs, together with others receptors for Ca2+, for sweet and amino acid taste compounds and for some pheromones, as well as for odorants in fish. Besides their transmembrane heptahelical domain responsible for G-protein activation, most of class III receptors possess a large extracellular domain responsible for ligand recognition. The recent resolution of the structure of this binding domain of one of these receptors, the mGlu1 receptor, together with the recent demonstration that these receptors are dimers, revealed an original mechanism of activation for these GPCRs. Such data open new possibilities to develop drugs aimed at modulating these receptors, and raised a number of interesting questions on the activation mechanism of other GPCRs.     

Facilitation of norepinephrine release from cerebral cortex is mediated by β2-adrenergic receptors

Facilitatory effects of prenalterol and albuterol (β1- and β2-selective adrenergic agonists, respectively) in the absence and presence of propranolol (a nonselective β-adrenergic antagonist), ICI 89,406 or ICI 118,551 (β1- and β2-selective adrenergic antagonists, respectively) on electrical stimulation-evoked release of ³H-NE from rat cerebral cortical slices were assessed. Albuterol (0.1 –100 nM) increased evoked release of ³H-NE from the cerebral cortical slices with greater potency than prenalterol (1 – 100 nM). The β2-adrenergic antagonist ICI 118,551 (1 nM) and propranolol (50 nM) abolished the facilitatory effects of albuterol (0.1 and 10 nM). In contrast, the βl-adrenergic antagonist ICI 89,406 (1 nM) did not alter the release-enhancing effect of albuterol. Prenalterol (10 and 100 nM)-induced facilitation of evoked release of ³H-NE was abolished by ICI 118,551; propranolol reduced the effect of 10 nM prenalterol and abolished that of 100 nM prenalterol. ICI 89,406 inhibited the effect of 100 nM prenalterol without altering that of 10 nM prenalterol. Basal release of ³H-NE was not altered by the drugs used in this study. These results suggest that facilitation of ³H-NE release induced by β-adrenergic agonists is mediated primarily by β2-adrenergic receptors.     

Transgenic rescue for characterizing orphan receptors: a review of δ2 glutamate receptor

Even though the genomes of several major species have been sequenced, many orphan receptors with unknown ligands and mechanisms of action remain in the CNS. The 2 glutamate receptor (GluR2) is one of such receptors expressed predominantly in the cerebellar Purkinje cells. On the basis of amino acid similarity, it belongs to ionotropic glutamate receptor (iGluR) family, which mediates fast excitatory neurotransmission in the mammalian CNS. Although its null-mutant mice show prominent motor discoordination, the mechanisms by which GluR2 participates in the cerebellar functions have been unclear. To gain insight into GluR2s mechanisms, we recently generated mice that express either a wild-type or a mutant GluR2 transgene, in which the conserved arginine in GluR2s N-terminal putative ligand-binding motif was disrupted. By breeding these transgenic mice onto a GluR2^–/– background, we obtained two transgenic rescue lines. Surprisingly, the mutant GluR2 transgene was as effective as the wild-type GluR2 in rescuing the GluR2-null mice. As the disrupted arginine residue is highly conserved from ancestral bacterial periplasmic amino acid-binding proteins to mammalian iGluRs, we propose that GluR2 may not require glutamate-like amino acids and may function in an unconventional manner. This transgenic rescue approach to investigating orphan receptors is a relatively easy but powerful method when a knockout mouse with a distinct phenotype is already available. The advantages and limitations of this approach, together with certain cautions in interpreting the resulting data, are discussed in this review.     

Effects of 2α-DHET and its optical isomers on muscarinic and nicotinic receptors

The agent 2α-(2′, 2′-disubstituted-2′-hydroxy-ethoxy) tropane (2α-DHET), its optical isomers and atropine were compared in their ability to inhibit specific [³H]QNB binding to muscarinic receptors of guinea pig ileum and to antagonize oxotremorine- and nicotine—induced contractions of isolated guinea pig ileum. A good correlation was observed between the affinities to muscarinic receptors and the antimuscarinic potencies in isolated guinea pig ileum. The binding data for 2α-DHET and its isomers were also consistent with their central and peripheral pharmacological activity in vivo. Compounds with 2′R configuration are more suitable to the stereostruture of the binding sites of muscarinic receptors than that of 2′S configuration.     

New immunology—immunology of pattern recognition receptors

Pattern recognition receptors (PRRs) have been found on all cells of the body—cells of the innate and adaptive immune systems, epithelial and endothelial cells, keratinocytes, etc. PRRs can recognize specific molecular structures of microorganisms as well as allergens and other substances. The interaction with ligands of foreign microorganisms activates PRRs, after which host cells start to produce cytokines both to specifically activate innate immunity and to control adaptive immune reactions. On the othe hand, no immune response develops against microorganisms of the normal microflora. Practically, the development of all immune responses is controlled by PRRs. These responses start in epithelial cells, skin cells, and vascular epithelial cells, which meet alien first. The immune system uses these cells to control the composition of normal microflora. Accordingly, the definition of immune system functions should be complemented by the regulation of body’s microflora in addition to the protection from alien and altered self.     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.