The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.

Shorr, Robert G. L.; Heald, Sarah L.; Jeffs, Peter W.; Lavin, T N; Strohsacker, Mark W.; Lefkowitz, Robert J.; Caron, Marc G.

doi:10.1073/pnas.79.9.2778

Cited by 72 publications

(29 citation statements)

References 15 publications

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“…(45 kDa) and comparable isoelectric points (pl) 5.8-6.2 [28,29] made it seem attractive to use native polyacrylamide gel in order to detect association between fliAR and &subunits in the presence of agonist. Non-denaturing polyacrylamide gels (4-5%) were prepared by the method of Hames [30] without a stacking gel.…”

mentioning

confidence: 99%

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Im¹,

Holzhöfer²,

Böttinger³

et al. 1988

FEBS Letters

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The role of the βγ‐subunits in the interaction of G‐proteins was examined with β1‐adrenoceptors purified from turkey erythrocytes and pure βγ‐subunits prepared from turkey erythrocytes and bovine brain. On a non‐denaturing polyacrylamide gel, the mobility of βγ‐subunits was increased when incubated with β1‐adrenoceptor and the β1‐adrenergic agonist l‐(−)‐isoproterenol, whereas on incubation with the antagonist l‐alprenolol the mobility was unchanged. Furthermore, the β1‐adrenoceptor was retarded on a Sephadex G‐50 column equilibrated with βγ‐subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated β1‐adrenoceptors with isolated βγ‐subunits of G‐proteins.

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mentioning

confidence: 99%

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Im¹,

Holzhöfer²,

Böttinger³

et al. 1988

FEBS Letters

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“…4) As indicated in Table 2, the reconstitution and fusion procedures described above can also be used with receptor prepared from rat lung and with receptor preparations of increasing purity. Table 2 by using BAR purified to apparent homogeneity (3)(4)(5).…”

Section: Resultsmentioning

confidence: 99%

“…In the initial phase, procedures were developed for the insertion of purified BARs [1][2][3][4][5][6]. Although attempts at exchanging digitonin in the receptor preparations with relatively low concentrations of dispersed phospholipids did result in a significant increase in the efficiency of reconstitution (Table 1, entry 7), it has not been possible to successfully couple this approach to a cell fusion procedure.…”

Section: Resultsmentioning

confidence: 99%

“…Second, they activate specific effectors such as enzymes and ion channels. The binding function of receptors has been probed by radioligand binding techniques (1,2), which have facilitated the purification of the binding macromolecules of the receptors (3)(4)(5). However, the "activating" function of such purified receptors has not generally been amenable to direct study.…”

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confidence: 99%

“…Purified frog erythrocyte membranes were prepared as described (15). The BAR was solubilized from erythrocyte plasma membranes (specific activity 1 pmol/mg of protein) and purified =100-fold by affinity chromatography on a Sepharose-alprenolol gel as described (4) but with some minor modifications (final specific activity =100 pmol/mg of protein). BAR activity was typically eluted with 0.025% digitonin/100 mM NaCl/10 mM Tris HCI, pH 7.4, containing 40-100 ,uM (±)-alprenolol rather than with a buffer containing 0.1% digitonin and 40 ,uM (±)-alprenolol.…”

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confidence: 99%

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Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.

Cerione¹,

Strulovici²,

Benovic³

et al. 1983

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

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The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified f8-adrenergic receptor preparations. Digitonin-solubilized J3-adrenergic receptors from frog erythrocytes or rat lung were purified -100-to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of 8-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted fi-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin El-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few ,3-adrenergic receptors and little or no catecholaminesensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2-to 10-fold) stimulation of the hybrid adenylate cyclase by the p3-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted fi-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the 8-adrenergic receptor prior to reconstitution. The successful coupling of a-purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure-function relationships.

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