AB5122 Sigma-AldrichAnti-Adrenergic Receptor Antibody, β 3

β(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against β(3)- and other β-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human β(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human β-adrenoceptor subtypes at near physiological levels (100-200 fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for β(3)-adrenoceptor labelling in cyto- or histochemistry studies.

TTo date, three ss-adrenoceptor (ss-AR) subtypes have been identified but only ss1- and ss2-ARs have been characterized in human lungs. Moreover, ss2-ARs physically interact with Cystic Fibrosis (CF) Transmembrane conductance Regulator (CFTR) through the Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) protein. ss3-ARs, which stimulate CFTR activity in transfected cells, have not been identified in human lungs. This study aimed firstly to characterize the presence of ss-AR subtypes, especially ss3-AR, in human bronchi, and secondly to compare their expression as well as NHERF1 in non-CF versus advanced CF lung samples. In human non-CF bronchi, ss1, ss2, ss3-ARs and NHERF1 transcripts and proteins were expressed mainly in bronchial epithelial cells. Those results were strengthened by the expression of ss1, ss2, ss3-AR in a human epithelial cell line, 16HBE14o-. All ss-AR subtypes stimulated CFTR activity. In CF bronchi, we demonstrate a ss1- and ss3-ARs overexpression and a NHERF1 and ss2-ARs underexpression. The origin of this protein remodelling (i.e. CFTR physical or functional absence, infection, inflammation, high adrenergic tone...) deserves further investigation. Nevertheless, these results evidence for the first time the presence of ss3-ARs in human bronchi and suggest them as a putative new pharmacological target in lung diseases where fluid homeostasis is altered. Furthermore, NHERF1 could be a new therapeutic target in CF patients to facilitate the trafficking of mutated CFTR to plasma membrane.

Preeclampsia, which complicates 3-8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of beta2- and beta2-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade of ADRB1 and ADRB2 by 0.1 microM propranolol but was significantly decreased by the blockade of ADRB1, ADRB2, and ADRB3 by 10 microM propranolol. Both SR 59119A and salbutamol were associated with a significant increase in cAMP production that was significantly inhibited by pretreatment with 0.1 microM propranolol only for salbutamol. SR 59119A-induced relaxation (E(max) = 28% +/- 5% vs. 45% +/- 4%, respectively) and cAMP production (2.7 +/- 0.5 vs. 4.9 +/- 0.4 pmol/mg of protein, respectively; P less than 0.01) were decreased in arteries obtained from preeclamptic compared to normotensive women. Both ADRB2 and ADRB3 transcripts were expressed at the same level between arteries from normotensive and preeclamptic women. Western blot analysis, however, revealed a decreased expression of the ADRB3 immunoreactive protein in arteries from preeclamptic compared to normotensive women. We suggest the presence of functional ADRB2 and ADRB3 in human placental arteries. Even if preeclampsia is associated with an impairment of the ADRB3 responsiveness, ADRB3 agonists may have future pharmaceutical implications in the management of pregnancy-related disorders.

Transcription-start sites for the mouse and human beta 3-adrenergic-receptor mRNA have been localized in a region comprised between 150 and 200 nucleotides 5' from the ATG translation-start codon. Motifs potentially implicated in heterologous regulation of beta 3-adrenergic-receptor expression by glucocorticoids and by beta-adrenergic agonists have been identified upstream from these cap sites. In mouse, a second mRNA initiation region is postulated to exist further upstream. Comparison of the nucleotide sequences of the 3' end of the human and mouse beta 3-adrenergic-receptor genes to those of the corresponding cDNA revealed that in contrast to beta 1 and beta 2 adrenergic receptors, the beta 3-adrenergic-receptor genes comprise several exons. A large exon (1.4 kb) encodes the first 402 and 388 amino-acid residues of the human and mouse beta 3 adrenergic receptor, respectively. In man, a second exon (700 bp) contains the sequence coding for the six carboxy-terminal residues of the receptor and the entire mRNA 3' untranslated region. In mouse, a second exon (68 bp) codes for the 12 carboxy-terminal residues of the receptor and a third exon contains the beta 3-adrenergic-receptor mRNA 3' untranslated region. The use of alternate acceptor splice sites generates two forms of exon 3 (600 bp and 700 bp), yielding two beta 3-adrenergic-receptor transcripts which are differentially expressed in white and brown adipose tissues. Human beta 3-adrenergic-receptor transcripts with different 3' untranslated regions are produced by continuation of transcription beyond termination signals. Together, our results suggest that utilization of alternate promoters and/or 3' untranslated regions may allow tissue-specific regulation of beta 3-adrenergic-receptors expression.

We report the molecular cloning of a beta 3-adrenergic receptor [beta 3-AR] cDNA from human brown adipose tissue. The cDNA-encoded protein is identical to the previously cloned beta 3-AR but with 6 additional amino acids at the C-terminus. The C-terminus is shared by the beta 3 receptors expressed in human neuroblastoma cells [SK-N-MC] [Mol. Pharmacol. 42 (1992) 964-970]. Furthermore, using a polymerase chain reaction strategy we have cloned and sequenced the beta 3-AR introns. Sequence analysis demonstrates that the human beta 3-AR gene comprises at least 3 exons and 2 introns and that the most abundant beta 3-AR transcripts encode a protein with an exon 3-derived C-terminus. Interestingly, although a similar organization has been found in rodent genes, the rat beta 3-AR transcripts encode a receptor with an exon 2-derived C-terminus.

DNA blot analysis of the cloned rat beta 3-adrenergic receptor gene revealed unexpected restriction enzyme cleavage sites that suggested the presence of one or more introns near the end of the coding block. This region of the rat gene was mapped and sequenced and was found to contain two introns. The first intron occurs 12 amino acids from the end of the coding block, as deduced by comparison with the beta 3 receptor cDNA. Sequence analysis of the first intron indicates that it might contain enhancer elements that could be important in the adipose tissue-specific expression of this gene. The mouse and human beta 3 receptor genes have been assumed to be intronless; however, these genes contain potential splice sites that are homologous to those present in the rat gene. The relevant regions of the mouse and human beta 3 receptor cDNAs were cloned and, by comparing them to the respective genomic sequences, it was concluded that these genes also contain one or more introns. Sequence analysis of the mouse and human beta 3 receptor cDNAs indicates that they code for proteins that are, respectively, 12 and 6 amino acids larger than previously deduced from genomic clones.

Since the classification of beta-adrenergic receptors (beta-ARs) into beta 1 and beta 2 subtypes, additional beta-ARs have been implicated in the control of various metabolic processes by catecholamines. A human gene has been isolated that encodes a third beta-AR, here referred to as the "beta 3-adrenergic receptor." Exposure of eukaryotic cells transfected with this gene to adrenaline or noradrenaline promotes the accumulation of adenosine 3',5'-monophosphate; only 2 of 11 classical beta-AR blockers efficiently inhibited this effect, whereas two others behaved as beta 3-AR agonists. The potency order of beta-AR agonists for the beta 3-AR correlates with their rank order for stimulating various metabolic processes in tissues where atypical adrenergic sites are thought to exist. In particular, novel beta-AR agonists having high thermogenic, antiobesity, and antidiabetic activities in animal models are among the most potent stimulators of the beta 3-AR.