Synthesis and characterization of CGP-12177-NBD: a fluorescent β-adrenergic receptor probe

Heithier, Helmuth; Jaeggi, Knut A.; Ward, Larry D.; Cantrill, R.C.; Helmreich, Ernst

doi:10.1016/0300-9084(88)90254-4

Cited by 18 publications

(14 citation statements)

References 13 publications

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“…Tota and Strader (1990) have characterized the binding site of a hamster /l,-adrenergic receptor expressed in baculovirus-infected Spodoptera frugiperdu insect cells with the antagonist carazolol as fluorescent probe. However, the results obtained with the fluorescent and more hydrophilic CGP-12177 derivative (Heithier et al, 1988) and the hydrophobic carazolol (Tota and were quite opposite; whereas the latter sensed a very hydrophobic environment, a more hydrophilic binding site was detected by the former probe. The third, large cytoplasmic loop, C3, and the cytoplasmic carboxy-terminal part differ among the members of this receptor family and parts of these structures could therefore be responsible for receptor-specific interactions with G-proteins (for additional information see also Dixon et al, 1988;Lefkowitz and Caron, 1988;Strader et al, 1989).…”

Section: Structural Properties Of Membrane Receptorsmentioning

confidence: 73%

“…This is mainly based on results from site-directed mutagenesis studies, deletion mutants and experiments with site-specific peptides (Dixon et al, 1987;Strader et al 1987aStrader et al , b, 1988Cotecchia et al, 1988Cotecchia et al, , 1990Fargin et al, 1988;Hamm et al, 1988;O'Dowd et al, 1988;Regan et al, 1988;Fraser, 1989;Palm et al, 1989Palm et al, , 1990Dohlman et al, 1990). Heithier et al (1988) have probed the environment of the ligand binding site with a fluorescent specific P-adrenergic antagonist which was environmentally sensitive. Tota and Strader (1990) have characterized the binding site of a hamster /l,-adrenergic receptor expressed in baculovirus-infected Spodoptera frugiperdu insect cells with the antagonist carazolol as fluorescent probe.…”

Section: Structural Properties Of Membrane Receptorsmentioning

confidence: 99%

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Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction

1991

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Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cellcybernetic aspects.Thanks to recombinant DNA technology, there has been a recent flood of structural information on membrane receptors, G-proteins and target enzymes [for reviews see Gilman

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Section: Structural Properties Of Membrane Receptorsmentioning

confidence: 73%

Section: Structural Properties Of Membrane Receptorsmentioning

confidence: 99%

Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction

1991

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“…All three subunits are heterogeneous, but the functional consequences of the considerable structural diversity are not yet clear (cf. Soege et al, 1991 Heithier et al, 1988); FITC, fluorescein isothiocyanate; TRM, tetramethylrhodamine maleimide. …”

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confidence: 99%

Subunit interactions of GTP‐binding proteins

Heithier

Frohlich

Dees

et al. 1992

European Journal of Biochemistry

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Pfeuffer, T. (1986) EMBO J. 5, 1509-15141. For this purpose, ao-and Bysubunits and trimeric Go purified from bovine brain, the fly-subunits from bovine rod outer segment membranes and the PI-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodaminmaleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of ao-and By-interactions, specific high-affinity binding interactions (& z 10 nM) and nonspecific low-affinity binding interactions (Kd z 1 pM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 pM guanosine 5'-O-[y-thio]triphosphate and 10 mM MgClz where only low-affinity binding interactions remained. Interactions between ao-and by-subunits from bovine brain or from bovine retina1 transducin did not differ much. The By-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated plreceptor reconstituted in lipid vesicles: Kd = 100 f 20 and 120 & 20 nM, respectively; however, Bysubunits from transducin appeared to bind more weakly to the P1-adrenoceptor than By-subunits from bovine brain. Separated purified homologous ao-and @subunits from bovine brain interfered mutually with each other in binding to the P1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein -protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Frohlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-1761 that /$subunits can bind to the nonactivated B1-adrenoceptor. GTP-binding proteins (G-proteins), which function in signal transmission pathways, are heterotrimers composed of three different subunits (a, /3 and y) which are coded by different genes. All three subunits are heterogeneous, but the functional consequences of the considerable structural diversity are not yet clear (cf. Soege et al., 1991 Heithier et al., 1988); FITC, fluorescein isothiocyanate; TRM, tetramethylrhodamine maleimide.

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“…1. Dependence of fluorescence intensity on the environment has been reported (19). For kinetic and drug-interaction studies partially purified rabbit skeletal muscle L-type Ca2+-channel protein at 0.003-0.008 mg/ml was incubated with 7.…”

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confidence: 99%

“…On the other hand, for neuronal, w-conotoxin GVIA-sensitive Ca2' channels, distribution and lateral mobility were recently investigated with a fluorescent toxin derivative (14), but data on L-type Ca2+ channels are unavailable. For localization studies at the cellular or subcellular level or on living cells fluorescent probes exist for the following receptors: nicotinic acetylcholine (15), dopamine (16,17), f3-adrenergic (18,19), benzodiazepine (20), glycine (21), insulin (22), glucagon (23), and vasopressin (24,25). Fluorescent probes were also developed for voltage-dependent Na' channels (26,27 METHODS Synthesi of the (4,4-Dlfluoro-5,7-dmethyl4-bora-3a,4a-daz)-3-(s-indacene)proplonlc Acid (DMBodipy)-and (4,4-Dfhoro-7-stry-4-bora-3a,4a-diaza)-3-(s-Dee)propioc Acid (STBodipy)-Labeled DHP Enatoers.…”

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confidence: 99%

In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.

Knaus¹,

Moshammer²,

Friedrich³

et al. 1992

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

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We have synthesized and charted fluorescently labeled dihydropyridines (DHos) as probes for L-type Ca2+ channels. Racemic as well as (+)-and (-)-1,4-dihydro-2,6-dlmethryl4-(2-trifluoromethylphenyl)-3,5-pyrdlhncrboxylic acid 2-(aminoethyl)ethyl ester hydrochlorides were coupled to boron dipyrromethane (Bodipy) derivatives. (4,4-Diluoro-5,7-dlmethyl-4-bora-3a,4a-diaza)-3-(s-andaceuepropn add (DMBodipy)-DHP and (4,4-difluoro-7-styryl-4bora-3a Radloligand Biding. Rabbit skeletal muscle transversetubule or guinea pig-cortex membranes were prepared as described (30). Rabbit skeletal muscle L-type Ca2+ channels were partially purified by solubilization and chromatography on wheat germ agglutinin-Sepharose (31). Fluorescent DHPs and other drugs were diluted and added in dimethyl sulfoxide

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Synthesis and characterization of CGP-12177-NBD: a fluorescent β-adrenergic receptor probe

Cited by 18 publications

References 13 publications

Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction

Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction

Subunit interactions of GTP‐binding proteins

In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.

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