Monoclonal antibodies to beta-adrenergic receptors: use in purification and molecular characterization of beta receptors.

Fraser, Claire M.; Venter, J. Craig

doi:10.1073/pnas.77.12.7034

Cited by 71 publications

(15 citation statements)

References 15 publications

(13 reference statements)

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“…In heart (Fig. 4, lanes F and G), no specific band could be seen, this is in agreement with data reporting that flAR detected in this organ are of the/3~ subtype [16,30].…”

Section: Distribution Of Immunoreactivity I N Hamster Tissuessupporting

confidence: 80%

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Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

1989

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We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung beta 2-adrenergic receptor (amino acids 16-31 and 174-189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified beta 2-adrenergic receptor in immunoblot conditions when used at a dilution of 1:500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as 125I-cyanopindolol labeled beta 2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the 125I-cyanopindolol binding domain of the beta 2-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified beta 2-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native beta 2-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing beta 2-adrenergic receptor-like molecules exhibited a neuronal phenotype.

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“…In heart (Fig. 4, lanes F and G), no specific band could be seen, this is in agreement with data reporting that flAR detected in this organ are of the/3~ subtype [16,30].…”

Section: Distribution Of Immunoreactivity I N Hamster Tissuessupporting

confidence: 80%

“…The specificity of the antisera was further confirmed by failure to react with purified rhodopsin (Fig. 4, lane H), a receptor-related molecule [47], or with hamster heart, a /3wAR-rich tissue [16], Fig. 4, lanes F and G.…”

Section: Molecules In Hamster Tissuesmentioning

confidence: 99%

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

1989

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“…Our studies over the past several years have yielded immunological and biochemical data suggesting substantial structural similarity among 81-, /82-, a1-and a2-adrenergic receptors and muscarinic cholinergic receptors (1)(2)(3). The extent of structural homology among these proteins became more apparent with the cloning and sequence analysis of genes encoding ,B-adrenergic receptors from human brain (4), hamster lung (5), and turkey erythrocytes (6) and muscarinic cholinergic receptors from porcine heart (7,8) and brain (9) (for review see ref.…”

mentioning

confidence: 99%

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Gocayne

Robinson²,

FitzGerald³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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152

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Two cDNA clones, X RHM-MF and X RHB-DAR, encoding the muscarinic cholinergic receptor and the 13-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart P-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac j8 receptor has 88.0% homology (92.5% with favored substitutions) with the human brain f3 receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and fi-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.Pharmacological and physiological classification of receptors has grouped muscarinic and nicotinic cholinergic receptors together in a class distinctly separate from the adrenergic receptors. Our studies over the past several years have yielded immunological and biochemical data suggesting substantial structural similarity among 81-, /82-, a1-and a2-adrenergic receptors and muscarinic cholinergic receptors (1-3). The extent of structural homology among these proteins became more apparent with the cloning and sequence analysis of genes encoding ,B-adrenergic receptors from human brain (4), hamster lung (5), and turkey erythrocytes (6) and muscarinic cholinergic receptors from porcine heart (7, 8) and brain (9) (for review see ref. 10).In a comprehensive review (11) concerning the evolution of adrenergic and cholinergic receptors, we postulated that the muscarinic cholinergic receptor has existed for >600 million years. Pharmacological and biochemical evidence suggests that the B-adrenergic receptor may have appeared later in evolution raising the possibility that it evolved as a result of gene duplication.We have cloned and determined the primary structure of the genes encoding ,B-adrenergic and muscarinic cholinergic receptors from rat heart.t To our knowledge, this is the first report on the cloning of a cardiac /3-adrenergic receptor, and these sequences have allowed the first direct sequence comparison between an adrenergic and a muscarinic cholinergic receptor from the same species and tissue. For these studies we utilized automated DNA sequence analysis (12, 13), a technique that will hav...

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“…Despite these successful attempts to produce polyclonal antibodies, only few monoclonal antibodies directed against ß-adrenergic receptors were described, whereas numerous antibodies against other receptors such as the nicotinic acetylcholine receptor or the epidermal growth factor (EGF) receptor are available. The first monoclonal antibodies directed against the ß-adrenergic receptor that were described, though promising, were rapidly lost after extinction of the hybridoma cell clones (6). More recently, our group produced monoclonal antibodies against the ß2-adrenergic receptor of the human epidermoid cell line A431 (7) and these were used to visualize ß-adrenergic receptors in different tissues (8,9).…”

Section: Introductionmentioning

confidence: 98%

A Monoclonal Antibody Directed Against the β₁-Adrenergic Receptor from Turkey Erythrocyte Membranes

Chapot¹,

Couraud²,

Schmutz³

et al. 1989

Hybridoma

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A monoclonal antibody was raised against the affinity purified beta 1-adrenergic receptor from turkey erythrocytes. This antibody, B120, of the IgG1 isotype, precipitates the photoaffinity-labeled purified receptor and the corresponding binding activity. The antibody recognizes the 42 kDa component of the receptor of erythrocyte membranes, after electrotransfer on nitrocellulose. B120 does not inhibit binding of dihydroalprenolol on membranes and has no effect on the activity of adenylate cyclase. Since specific immunofluorescence was detected only after ethanol treatment of the erythrocytes, the epitope recognized by B120 appears not to be accessible at the cell surface.

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Monoclonal antibodies to beta-adrenergic receptors: use in purification and molecular characterization of beta receptors.

Cited by 71 publications

References 15 publications

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

A Monoclonal Antibody Directed Against the β₁-Adrenergic Receptor from Turkey Erythrocyte Membranes

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Monoclonal antibodies to beta-adrenergic receptors: use in purification and molecular characterization of beta receptors.

Cited by 71 publications

References 15 publications

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

A Monoclonal Antibody Directed Against the β1-Adrenergic Receptor from Turkey Erythrocyte Membranes

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A Monoclonal Antibody Directed Against the β₁-Adrenergic Receptor from Turkey Erythrocyte Membranes