Transferability of thermostabilizing mutations between β-adrenergic receptors

Abstract

In previous work we described six point mutations that thermostabilised the turkey β₁-adrenergic receptor (tβ₁AR). The thermostable mutant, tβ₁AR-m23, had an apparent T_m 21°C higher than the native protein when solubilized in dodecylmaltoside (DDM) and, in addition, was significantly more stable in short chain detergents, which allowed its crystallization and structure determination. Identification of thermostabilizing mutations in tβ₁AR was performed by systematic mutagenesis followed by expressing and assaying each of the 318 mutants for their thermostability. This is time-consuming, so to facilitate studies on related receptors, we have studied the transferability of these mutations to the human adrenergic receptors, hβ₁AR and hβ₂AR, which have, respectively, 76% and 59% sequence identity to tβ₂AR, excluding the N- and C-termini. Thermostability assays revealed that hβ₁AR was much more unstable than tβ₂AR, whereas hβ₂AR was more stable than tβ₁AR. Addition of the 6 thermostabilizing mutations in tβ₂AR-m23 into both hβ₂AR and hβ₂AR increased their apparent T_ms by 17°C and 11°C, respectively. In addition, the mutations affected the global conformation of the human receptors so that they were predominantly in the antagonist bound form, as was originally observed for tβ₂AR-m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable.     

Biological maturation and β-adrenergic effectors: development of β-adrenergic receptors in rabbit heart

Summary The -adrenergic receptor, transduction processes and catalytic activity of the adenylate cyclase enzyme complex have been investigated in rabbit heart at different stages of biological maturation. The binding of [³H]-dihydroalprenolol to a washed membrane preparation isolated from rabbit ventricular muscle was used to characterize -adrenergic receptors. Significant age-related differences were noted in -receptor affinity (K_d) and density (RD) of neonatal and adult animals; the adult K_d was 3.7-fold greater and the RD 2-fold higher than the neonates. No significant differences in these parameters were detected among the 27-day old fetus and the 1- and 7-day old neonates. Age-dependent differences in agonist isoproterenol affinity for the receptor were not observed in contrast to the significant changes in antagonist (DHA) affinity.Age-related changes in receptor affinity were also quantitated by determining the inhibitory potency of alprenolol on isoproterenol stimulated adenylate cyclase enzyme activity. A decreased affinity of the -adrenergic receptor for alprenolol in the adult heart was indicated by a 3.7-fold greater K_i for the adult than the 1-day old neonate. Ontogenic variations in the coupling efficiency between the receptor and catalytic components of the adenylate cyclase complex were also evaluated. The K_d of the -adrenergic receptor for isoproterenol and the EC50 for adenylate cyclase stimulation were determined under similar conditions. The corresponding coupling index (K_d/EC50) was found to be 2.4-fold greater in the 1-day old neonate than adult, suggesting that for a given percentage increase in adenylate cyclase activity, a lower percentage of -adrenergic receptor sites need be occupied in the neonate. These data extend previous studies (29) and indicate all components of the rabbit heart adenylate cyclase enzyme complex (i.e., the -adrenergic receptor, the GTP-dependent transduction event, and the catalytic subunit) exhibit significant developmental changes.     

Biased agonism at β-adrenergic receptors

The β-adrenergic receptors (βARs) include three subtypes, β₁, β₂ and β₃. These receptors are widely expressed and regulate numerous physiological processes including cardiovascular and metabolic functions and airway tone. The βARs are also important targets in the treatment of many diseases including hypertension, heart failure and asthma. In some cases, the use of current βAR ligands to treat a disease is suboptimal and can lead to severe side effects. One strategy to potentially improve such treatments is the development of biased agonists that selectively regulate a subset of βAR signaling pathways and responses. Here we discuss the compounds identified to date that preferentially activate a G_s- or β-arrestin-mediated signaling pathway through βARs. Mechanistic insight on how these compounds bias signaling sheds light on the potential development of even more selective compounds that should have increased utility in treating disease.     

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.     

Molecular basis of ligand dissociation in β-adrenergic receptors

The important and diverse biological functions of β-adrenergic receptors (βARs) have promoted the search for compounds to stimulate or inhibit their activity. In this regard, unraveling the molecular basis of ligand binding/unbinding events is essential to understand the pharmacological properties of these G protein-coupled receptors. In this study, we use the steered molecular dynamics simulation method to describe, in atomic detail, the unbinding process of two inverse agonists, which have been recently co-crystallized with β(1) and β(2)ARs subtypes, along four different channels. Our results indicate that this type of compounds likely accesses the orthosteric binding site of βARs from the extracellular water environment. Importantly, reconstruction of forces and energies from the simulations of the dissociation process suggests, for the first time, the presence of secondary binding sites located in the extracellular loops 2 and 3 and transmembrane helix 7, where ligands are transiently retained by electrostatic and Van der Waals interactions. Comparison of the residues that form these new transient allosteric binding sites in both βARs subtypes reveals the importance of non-conserved electrostatic interactions as well as conserved aromatic contacts in the early steps of the binding process.     

Increased number of β-adrenergic receptors in the hypertrophied myocardium

Development of cardiac hypertrophy is associated with depletion of endogenous catecholamine stores and increased inotropic response to exogenous catecholamines. A biochemical basis for these changes is provided by the observation that the number of cardiac β-adrenergic receptors—as reflected in specific [³H]dihydroalprenolol binding—is increased in hypertrophy without a change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. This change in β-receptor numbers may be an important adaptive mechanism for preserving the contractile performance of the hypertrophied myocardium.     

Development of covalent antagonists for β1- and β2-adrenergic receptors

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β₂-adrenergic receptor (β₂AR^H2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β₁- and β₂ receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β₁AR^I2.64C and β₂AR^H2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β₂AR^H2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β₂AR^H2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β₁AR^I2.64C and β₂AR^H2.64C mutant while retaining a 41-fold selectivity for the β₁AR wild type over β₂AR. These compounds may serve as valuable molecular tools for studying β₁/β₂ subtype selectivity or investigations on GPCR trafficking and dimerization.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

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

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

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.     

Simultaneous assay of muscarinic and β-adrenergic receptors using a double isotope technique

Muscarinic receptor and β-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [³H]-L-quinuclidinyl benzilate and [¹²⁵I]-(?)iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,l-propranolol on [³H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of l-quinuclidinyl benzilate on [¹²⁵I]-(?)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.     

Regulation of a presynaptic adenylate cyclase from bovine cerebellum by β-adrenergic receptors

Intact crude synaptosomes from bovine cerebellum contain, in addition to an externally accessible (postsynaptic) adenylate cyclase, an enzyme with its catalytic center oriented towards the inside of the synaptosome (presynaptic adenylate cyclase). This is demonstrated by the unmasking of latent adenylate cyclase activity by Triton X-100. Furthermore, intact crude synaptosomes can synthesize cyclic AMP from adenine. This synthesis takes place inside the synaptosome as the postsynaptic adenylate cyclase is inactive in the Krebs-Ringer buffer. Presynaptic adenylate cyclase activity is not influenced by depolarization, as shown by [³H]adenine pulse-labeling, but is stimulated by (?)-norepinephrine and (?)-isoproterenol. (±)-Propranolol inhibits this stimulation whereas phentolamine has no effect, suggesting the presence of a β-adrenergic receptor-coupled presynaptic adenylate cyclase.     

Direct insertion and fluorescence studies of rhodamine-labeled β-adrenergic receptors in cell membranes

Summary Previous studies utilizing the fluorescence of propanolol as a probe for the beta-adrenergic receptor showed that this receptor is motionally constrained. To further study the betaadrenergic receptorin situ we have reinserted rhodamine-labeled beta-receptors into cell membranes. This report presents documentation of their insertion and physiologic viability. Beta-receptors were purified by affinity chromatography (10,000-fold), then fluorescently labeled with tetramethyl rhodamine isothiocyanate, repurified (55,000-fold) and incubated with rat pancreatic islet cells. The binding of³H-dihydroalprenolol by these cells was increased from aB _max of 168±2 to 309±20 fmol/mg protein with no change inK _d . Various treatments which remove peripheral membrane proteins, e.g. NaOH, lithium diiodosalicylate, and trypsinization, did not alter binding by the cells with inserted receptors indicating that the receptors inserted into cell membranes. In islet cells treated with Koshland's reagent I, beta-adrenergic binding was completely abolished, but following incubation with isolated beta-receptors, the cells bound beta-adrenergic radioligand with aB _max of 100 fmol/mg protein, indicating functionality on the part of the inserted receptors. Furthermore, insertion of isolated receptors into frog erythrocytes resulted in increased production of cAMP in response to added isoproterenol. In pancreatic islet cells, incubation with labeled receptors caused the fluorescence to shift in wavelength with increased intensity indicating a shift from an aqueous to a lipid environment, probably into the membrane. Using fluorescence (P), it was found that the inserted receptors became motionally constrained to aP of 0.38 (limitingP _o=0.42) during the first 15 min, remaining so for at least 2 hr. Colchicine (5 g/ml) caused a decrease inP to 0.18 indicating release of constraint. Isoproterenol (10^–5M) caused a rapid decrease toP=0.15. This effect was blocked by propranolol. Propranolol itself (10^–5M) had no effect. These results indicate that the labeled receptors rapidly insert into cell membranes and also support the view that agonist activation of the receptor causes an increase in receptor mobility, presumably due to release of constraint from cytoskeleton elements.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

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.     

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.     

Regulation of β-adrenergic receptor function

G protein-coupled receptors are the largest family of cell surface receptors regulating multiple cellular processes. β-adrenergic receptor (βAR) is a prototypical member of GPCR family and has been one of the most well studied receptors in determining regulation of receptor function. Agonist activation of βAR leads to conformational change resulting in coupling to G protein generating cAMP as secondary messenger. The activated βAR is phosphorylated resulting in binding of β-arrestin that physically interdicts further G protein coupling leading to receptor desensitization. The phosphorylated βAR is internalized and undergoes resensitization by dephosphorylation mediated by protein phosphatase 2A in the early endosomes. Although desensitization and resensitization are two sides of the same coin maintaining the homeostatic functioning of the receptor, significant interest has revolved around understanding mechanisms of receptor desensitization while little is known about resensitization. In our current review we provide an overview on regulation of βAR function with a special emphasis on receptor resensitization and its functional relevance in the context of fine tuning receptor signaling.