The fat cell beta-adrenergic receptor. Purification and characterization of a mammalian beta 1-adrenergic receptor.

Cubero, Ana; Malbon, Craig C.

doi:10.1016/s0021-9258(17)43608-8

Cited by 59 publications

(8 citation statements)

References 35 publications

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“…The Mr of purified human /8-adrenergic receptors on SDS/polyacrylamide gels is similar to that of purified /l.-adrenergic receptor from rat fat-cells (Mr 67000) (Cubero & Malbon, 1984) and /32-adrenergic receptors of guinea-pig lung (Mr 67000) (Drake & Perkins, 1984), hamster lung (Mr 64000) (Benovic et al, 1984), rat liver (Mr 67000) (Graziano et al, 1985) and mouse lymphoma S49 cells (Mr 67000) (George & Malbon, 1985). Autoradiograms of /,-adrenergic receptors identified by photoaffinity labelling of basal membranes prepared from human placenta by [125I]iodoazidobenzylpindolol also revealed a predominant Mr-65000-67000 peptide, in addition to a lesser quantity of Mr-55000 and a novel Mr-76000 species (Bahouth et al, 1986).…”

Section: Discussionmentioning

confidence: 57%

“…,-Adrenergic receptors are subdivided into two distinct subclasses, termed f,1and f2-, based on pharmacological criteria (Lands et al, 1967). Unlike the differences observed between amphibian f82and avian fl1-adrenergic receptors (Stiles et al, 1984a), for mammalian fl1and fl2-receptors many structural features appear in common (Cubero & Malbon, 1984;Benovic et al, 1984;Graziano et al, 1985;George & Malbon, 1985). Both receptor subtypes appear to be glycoproteins (Stiles et al, 1984a;Benovic et al, 1984;George et al, 1986) and contain intramolecular disulphide bridges Moxham et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

“…A sample (0.5 ml) of DEAE-Sephacel eluate was subjected to steric-exclusion h.p.l.c. as described by Cubero & Malbon (1984). The mobile phase was composed of 0.2 M-NaCl, 0.1 M-Na2HPO4/NaH2PO4 (pH 7.0), 1 mM-EDTA, 0.1 mM-PMSF and 0.1 % digitonin.…”

Section: Introductionmentioning

confidence: 99%

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Human β-adrenergic receptors. Simultaneous purification of β1- and β2-adrenergic-receptor peptides

Bahouth

Malbon

1987

Biochemical Journal

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Beta-Adrenergic receptors from basal membranes of human placenta were purified from digitonin extracts by sequential rounds of affinity chromatography, hydrophobic chromatography, ion-exchange chromatography and steric-exclusion h.p.l.c. Basal membranes display both beta 1- and beta 2-adrenergic receptors, in the ratio 65:35. Affinity chromatography, hydrophobic chromatography on heptylamine-Sepharose and ion-exchange chromatography on DEAE-Sephacel removed most of the contaminating proteins, and final purification of the receptor to apparent homogeneity was achieved by steric-exclusion h.p.l.c. The purified receptors showed Mr 67000 on SDS/polyacrylamide-gel electrophoresis under reducing conditions. Specific binding of radioligand to the purified beta-adrenergic receptors displayed stereoselectivity, and the agonist competition profiles demonstrated the presence of both beta 1- and beta 2-receptors. By using the subtype-selective ligands CGP-20712A (beta 1-selective) and ICI-118,551 (beta 2-selective), the purified Mr-67000 species was shown to be composed of equivalent amounts of beta 1- and beta 2-adrenergic receptors. Affinity chromatography on Sepharose-alprenolol and sequential elution with 1 microM-CGP-20712A followed by 100 microM(-)-alprenolol permitted beta 1-adrenergic receptors to be resolved from the mixture of beta 1-/beta 2-adrenergic receptors. The pharmacologically distinct human beta 1 and beta 2-adrenergic receptors are shown to be structurally very similar peptides.

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Section: Discussionmentioning

confidence: 57%

Section: Introductionmentioning

confidence: 99%

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Human β-adrenergic receptors. Simultaneous purification of β1- and β2-adrenergic-receptor peptides

Bahouth

Malbon

1987

Biochemical Journal

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“…Radioligand Binding Assay. Measurements of /3-adrenergic receptors in fat cell membranes, digitonin extracts prepared from these membranes, and purified preparations of receptor were performed with the high-affinity, /3-adrenergic antagonist (-)-[3H]dihydroalprenolol as described previously (Malbon et al, 1978;Cubero & Malbon, 1984). /3-Mercaptoethanol, dithiothreitol, and jV-ethylmaleimide when employed were added directly to the incubation mixture containing radioligand and receptor preparations.…”

Section: Methodsmentioning

confidence: 99%

“…The specific binding of radioligands to 3-adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. 3-Adrenergic receptor purified from rat fat cells and treated with 3-mercaptoethanol (10%) and then subjected to gel electrophoresis in the presence of sodium dodecyl sulfate migrates as a Mr 67 000 peptide [Cubero, A., & Malbon, C. C. (1984) J. Biol.…”

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Fat cell .beta.1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding

Moxham¹,

Malbon²

1985

Biochemistry

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Agents that react chemically with sulfhydryl groups of proteins modify the response of adenylate cyclase to stimulation by beta-adrenergic agonists. N-Ethylmaleimide, an agent that alkylates sulfhydryl groups, inactivates both the catalytic moiety of adenylate cyclase and the stimulatory, regulatory guanine nucleotide binding protein Ns of rat fat cells but fails to affect binding of antagonists to the beta-adrenergic receptor [Malbon, C. C., Graziano, M. P., & Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260]. Treating membranes of rat fat cells with dithiothreitol or beta-mercaptoethanol, agents that reduce disulfide bridges of proteins, results in a loss of binding of beta-adrenergic radioligands to the receptor. The specific binding of radioligands to beta-adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. beta-Adrenergic receptor purified from rat fat cells and treated with beta-mercaptoethanol (10%) and then subjected to gel electrophoresis in the presence of sodium dodecyl sulfate migrates as a Mr 67 000 peptide [Cubero, A., & Malbon, C. C. (1984) J. Biol. Chem. 259, 1344-1350]. In the absence of disulfide bridge reducing agents, however, the purified receptor exhibits greater electrophoretic mobility, migrating as a peptide with Mr 54 000. Treating the native form of the purified receptor with beta-mercaptoethanol (0.1-10%) or dithiothreitol (0.1-10 mM) decreases the ability of the receptor to bind beta-adrenergic ligands, decreases the electrophoretic mobility of the receptor, and results in receptor peptides migrating with molecular weight ranging from 54 000 to 67 000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

Bar-Sinai¹,

Aldouby²,

Chorev³

et al. 1986

The EMBO Journal

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We have recently reported that the highly potent beta‐adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM‐CYP) irreversibly blocks the turkey erythrocyte beta‐adrenoceptor binding site by combining with a receptor‐associated non‐protein component. In this communication, we report: lipid labelling is inhibited by beta 1‐adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1‐adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM‐CYP‐blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM‐CYP‐glycolipid with restoration of the binding capacity of the beta 1‐adrenoceptor protein; the tightly associated [125I]BAM‐CYP‐labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor‐mediated signal transduction is discussed in view of these findings and in view of data from the literature.

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The fat cell beta-adrenergic receptor. Purification and characterization of a mammalian beta 1-adrenergic receptor.

Cited by 59 publications

References 35 publications

Human β-adrenergic receptors. Simultaneous purification of β1- and β2-adrenergic-receptor peptides

Human β-adrenergic receptors. Simultaneous purification of β1- and β2-adrenergic-receptor peptides

Fat cell .beta.1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

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