Probing human β1- and β2-adrenoceptors with domain-specific fusion protein antibodies

doi:10.1016/S0014-2999(96)00654-1

European Journal of Pharmacology

Volume 316, Issue 1, 28 November 1996, Pages 111-121

https://doi.org/10.1016/S0014-2999(96)00654-1 Get rights and content

Abstract

In order to generate antibodies suitable for immunological studies on β-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human β₁- or β₂-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human β₁- or β₂-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the β₁-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the ³H-labelled antagonist (−)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-³H]benzimidazol-2-one ([³H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [³H]CGP 12 177. Affinity purified antibodies were used for detecting the β₁- or the β₂-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying β-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

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Diagnostic and therapeutic aspects of β<inf>1</inf>-adrenergic receptor autoantibodies in human heart disease
2014, Autoimmunity Reviews
Citation Excerpt :
Similar effects were demonstrated for antibodies produced in rodents against peptides corresponding to the second extracellular β1AR loop, which cross-react with the native β1AR [35,38,39]. Moreover, such immunisations induced left ventricular dilation and dysfunction [9,10,40] among other effects compatible with chronic cardiac dysfunction [11–13]. These pathological effects were reversible upon removal of the antibodies [41], transferable via serum transfusions [10,13,42,43], and partially abrogated by β1AR antagonists [44] or synthetic mimics of the receptor epitope [45,46].
Growing evidence indicates a cardio-pathogenic role of autoantibodies against β₁-adrenergic receptors (β₁AR). In particular autoantibodies stimulating β₁AR-mediated cAMP-production (i.e. agonistic β₁AR autoantibodies) play a paramount role in chronic heart failure. When induced by immunisation, such autoantibodies cause heart failure in rodents; when present in patients they negatively affect survival in heart failure. However, the true prevalence and clinical impact of agonistic β₁AR autoantibodies in human heart disease are still unclear, as are the events triggering their production, and the inter-relationship between autoantibody level and disease activity. β₁AR autoantibodies can be removed by extracorporeal absorption or neutralised by systemic administration of synthetic epitope mimics. Only patients bearing agonistic β₁AR autoantibodies in their bloodstream will benefit from these approaches. Therefore, reliable detection of agonistic β₁AR autoantibodies is a key pre-requisite for the future implementation of these strategies. β₁AR autoantibodies impact on conformation and down-stream signalling of the receptor by binding a conformational epitope, which is poorly represented by synthetic mimics and readily destroyed by fixation. Consequently, β₁AR autoantibodies can reliably be detected only by assays utilising the native β₁AR as a test antigen. To provide a sufficient basis for diagnostic predictions or therapeutic decisions, one must also determine whether β₁AR autoantibodies stimulate the receptor, which again requires native, cell-based reporter systems. Translation of these procedures into versatile diagnostic tests fitting the requirements of general health care is a challenge for future development. Here, we will review the state of diagnostic and therapeutic efforts in the field of β₁AR-directed autoimmunity, thereby aiming to furnish a conceptual frame for the further development of novel, more reliable diagnostic tools and more specific antibody-targeted therapeutic concepts.
A Novel Fluorescence Method for the Rapid Detection of Functional β<inf>1</inf>-Adrenergic Receptor Autoantibodies in Heart Failure
2007, Journal of the American College of Cardiology
This study sought to develop a rapid method for the detection of activating autoantibodies directed against the β₁-adrenoceptor (anti-β₁-Abs) in patients with heart failure.
The anti-β₁-Abs are supposed to play a pathophysiological role in heart failure. However, there is no reliable method for their detection. With a complex screening strategy (enzyme-linked immunosorbent assay, immunofluorescence, cyclic adenosine monophosphate [cAMP]–radioimmunoassay) we have previously identified antibodies targeting the second extracellular β₁-receptor loop (anti-β₁-EC_II) in 13% of patients with ischemic cardiomyopathy (ICM) and in 26% with dilated cardiomyopathy (DCM).
To detect anti-β₁-Abs, we measured β₁-receptor–mediated increases in intracellular cAMP by fluorescence resonance energy transfer using a highly sensitive cAMP sensor (Epac1-based fluorescent cAMP sensor).
The immunoglobulin G (IgG) prepared from 77 previously antibody-typed patients (22 ICM/55 DCM) and 50 matched control patients was analyzed. The IgG from all 22 previously anti-β₁-EC_II–positive patients (5 ICM/17 DCM) induced a marked cAMP increase, indicating receptor activation (49.8 ± 4.2% of maximal isoproterenol-induced signal). The IgG from control patients and 32 previously anti-β₁-EC_II–negative patients (17 ICM/15 DCM) did not significantly affect cAMP. Surprisingly, our technology detected anti-β₁-Abs in 23 DCM patients formerly judged antibody-negative, but their cAMP signals were generally lower (31.3 ± 6.8%) than in the previous group. “Low”-activator anti-β₁-Abs were blocked preferentially by peptides corresponding to the first, and “high”-activator anti-β₁-Abs by peptides corresponding to the second β₁-extracellular loop. Beta-blockers alone failed to fully prevent anti-β₁-EC_II–induced receptor activation, which could be achieved, however, by the addition of β₁-EC_IIpeptides.
Our novel method of detecting anti-β₁-Abs proved to be fast and highly sensitive. It also revealed an insufficient ability of beta-blockers to prevent anti-β₁-EC_II–induced receptor activation, which opens new venues for the research on anti-β₁-Abs and eventual treatment options in heart failure.
Beta 1-adrenergic receptor-directed autoimmunity as a cause of dilated cardiomyopathy in rats
2006, International Journal of Cardiology
Progressive cardiac dilatation and pump failure of unknown etiology has been termed idiopathic dilated cardiomyopathy (DCM). During recent years a large body of data has accumulated indicating that functionally active antibodies or autoantibodies being able to recognize and to stimulate the cardiac β₁-adrenergic receptor (anti-β₁-AR) may play an important role in the initiation and/or clinical course of DCM. Recent experiments in rats even point towards a cause-and-effect relation between stimulatory anti-β₁-AR antibodies and DCM. Immunization of rats against the second extracellular loop of the human β₁-adrenergic receptor (100% sequence-identity between human and rat) resulted in both development of stimulatory anti-β₁-AR antibodies and development of progressive cardiac dilatation and dysfunction. Isogenic transfer of stimulatory anti-β₁-AR from cardiomyopathic into healthy inbread animals reproduced the disease, hence providing conclusive proof for a β₁-receptor-directed autoimmune attack as a possible cause of cardiomyopathy. This kind of cardiomyopathy is now referred to as anti-β₁-AR-induced dilated immune-cardiomyopathy (DiCM).
The following article reviews recent evidence obtained from experimental animal-models implying a significant role of the cardiac β₁-adrenergic receptor as a pathophysiologically and clinically relevant autoantigen also in human DCM.
β<inf>1</inf>-Adrenergic receptor function, autoimmunity, and pathogenesis of dilated cardiomyopathy
2006, Trends in Cardiovascular Medicine
Dilated cardiomyopathy (DCM) is a heart disease characterized by progressive depression of cardiac function and left ventricular dilatation of unknown etiology in the absence of coronary artery disease. Genetic causes and cardiotoxic substances account for about one third of the DCM cases, but the etiology of the remaining 60% to 70% is still unclear. Over the past two decades, evidence has accumulated continuously that functionally active antibodies or autoantibodies targeting cardiac β₁-adrenergic receptors (anti-β₁-AR antibodies) may play an important role in the initiation and/or clinical course of DCM. Recent experiments in rats indicate that such antibodies can actually cause DCM. This article reviews current knowledge and recent experimental and clinical findings focusing on the role of the β₁-adrenergic receptor as a self-antigen in the pathogenesis of DCM.
Immunofluorescent imaging of β<inf>1</inf>- and β<inf>2</inf>-adrenergic receptors in rat kidney
2001, Kidney International
Citation Excerpt :
Antibodies against the β2-AR (anti–β2-AR) labeled a protein band of slightly higher mobility (Figure 1, lanes 6 through 8), which is also plausible in the light of the (slightly shorter) amino acid sequence of the rat β2-AR15 when taking protein glycosylation into account. In this context, it should be noted that β-AR are known to be less glycosylated when expressed in Sf9 insect cells13,18, giving protein bands with apparent sizes of 51 and 47 kD for the recombinant human β1- and β2-AR, respectively (Figure 1, lanes 4 and 9). These data indicated that these antibodies did recognize rat β1- and β2-AR, and thus they appeared to be suitable for an immunohistological study on rat kidney.
Immunofluorescent imaging of β₁- and β₂-adrenergic receptors in rat kidney.
β-Adrenergic receptors (β-ARs) are known to participate in the regulation of glomerular filtration, NaCl reabsorption, acid-base balance, and renin secretion; however, the precise histologic localization of β-AR at putative signaling sites involved in these processes remains an open issue.
We used a set of subtype-specific rabbit antibodies to visualize β₁- and β₂-AR in rat kidney by immunohistochemistry and specified cells and segments of the nephron thought to be regulated by catecholamines. In addition, the relative proportion of β-AR subtypes in cortical and medullary portions of rat kidney was determined by Western blotting and by competing [¹²⁵I]-cyanopindolol binding with the β₁- or β₂-selective antagonists bisoprolol or ICI 118,551, respectively.
Immunoreactivity for β₁-AR was found in mesangial cells, juxtaglomerular granular cells, the macula densa epithelium, proximal and distal tubular segments, and acid-secreting type A intercalated cells of the cortical and medullary collecting ducts. Immunoreactivity for β₂-AR was predominantly localized in the apical and subapical compartment of proximal and, to a lesser extent, distal tubular epithelia (suggesting interactions with luminal fluid catecholamines). Both subtypes were dense in the membranes of smooth muscle cells from renal arteries. Concordant data were obtained by radioligand binding and immunoblotting of membranes prepared from cortical and medullary portions of the kidney.
Our data provide an immunohistochemical basis for the cellular targets of β-adrenergic regulation of renal function. Moreover, they could help to devise therapeutic strategies directed at renal β-ARs.
Astrocyte β<inf>1</inf>-adrenergic receptor immunoreactivity and agonist induced increases in [Ca<sup>2+</sup>](i): Differential results indicative of a modified membrane receptor
2000, Life Sciences
Antibodies against the C-terminus of the β₁-adrenergic receptor were used for staining cultured astrocytes from the rat cerebral cortex. Immunoreactivity was found to be localized exclusively to an intracellular organelle structure similar to the Golgi complex, with no staining of the plasma membrane. The astrocytes stained positive with BODIPY CGP 12177, a FITC-conjugated β-adrenergic receptor agonist, and this staining was blocked by the β₁-adrenergic antagonist atenolol, indicating that these receptors are expressed on the surface of the astrocytes. The presence of functional plasma membrane β₁-adrenergic receptors was further verified using microspectrofluorometry for measurements of intracellular calcium changes upon β-adrenergic agonist stimulation. Intracellular immunoreactivity confined to the organelles was also found in astrocytes from mixed astroglial-neuronal cultures. In contrast, the neurons in these cultures showed a strong labeling of the cell bodies by the β₁-adrenergic receptor antibodies. Thus, the β₁-adrenergic receptor antibody, which stains the cell bodies of the neurons, recognizes the astroglial receptors only intracellularly, although functional β₁-adrenergic receptors are present on the astroglial surface. Taken together, these data suggest that the β₁-adrenergic receptors observed intracellularly might be processed on their passage to the surface to a modified form of the final plasma membrane receptor, which is not recognized by the antibodies.

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Probing human β1- and β2-adrenoceptors with domain-specific fusion protein antibodies

Abstract

Trends Pharmacol. Sci.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

Trends Neurosci.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry

J. Neurosci.

A monoclonal antibody directed against the beta 1-adrenergic receptor from turkey erythrocyte membranes

Hybridoma

Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli

Eur. J. Biochem.

Probing human β₁- and β₂-adrenoceptors with domain-specific fusion protein antibodies