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

Synthesis and Properties of Fluorescent .beta.-Adrenoceptor Ligands

“…Finally, the characteristics of CGP 12177 binding to recombinant human /32-adrenoceptors expressed in Sf9 cells are similar to those reported from human cell lines constitutively expressing ^-adrenoceptors (Boege et al, 1988;Heithier et al, 1988a;Staehelin et al, 1983). Comparing the Kn values obtained by displacement of radioactive [3H]CGP 12177 bound to /3-adrenoceptors of Sf9 cells by fluorescent BODIPY-CGP (Figure 2a) with those obtained by fluorometry of BODIPY-CGP bound to Sf9 cell membranes (Figure 2b) and with data obtained by direct photon counting of BODIPY-CGP bound to single intact cells (Figure 2c,d) shows that the favorable ligand binding properties of CGP 12177 are not compromised by the derivatization with BODIPY.…”

“…For example, both would be expected to react with a common sequencerecognizing antibody, but only the former would be expected to bind the ligand with specificity and high affinity (von Zastrow & Kobilka, 1992). Therefore, we have made efforts to synthesize specific and tight-binding fluorescent /3-adrenergic ligands (Heithier et al, 1988a;Henis et al, 1982). In previous work (Henis et al, 1982), NBD-alprenolol was used because CGP 12177 was not yet available.…”

Synthesis and Properties of Fluorescent .beta.-Adrenoceptor Ligands

“…Finally, the characteristics of CGP 12177 binding to recombinant human /32-adrenoceptors expressed in Sf9 cells are similar to those reported from human cell lines constitutively expressing ^-adrenoceptors (Boege et al, 1988;Heithier et al, 1988a;Staehelin et al, 1983). Comparing the Kn values obtained by displacement of radioactive [3H]CGP 12177 bound to /3-adrenoceptors of Sf9 cells by fluorescent BODIPY-CGP (Figure 2a) with those obtained by fluorometry of BODIPY-CGP bound to Sf9 cell membranes (Figure 2b) and with data obtained by direct photon counting of BODIPY-CGP bound to single intact cells (Figure 2c,d) shows that the favorable ligand binding properties of CGP 12177 are not compromised by the derivatization with BODIPY.…”

“…For example, both would be expected to react with a common sequencerecognizing antibody, but only the former would be expected to bind the ligand with specificity and high affinity (von Zastrow & Kobilka, 1992). Therefore, we have made efforts to synthesize specific and tight-binding fluorescent /3-adrenergic ligands (Heithier et al, 1988a;Henis et al, 1982). In previous work (Henis et al, 1982), NBD-alprenolol was used because CGP 12177 was not yet available.…”

Dynamics of β2-Adrenergic Receptor−Ligand Complexes on Living Cells

“…Competition experiments with the β 1 /β 2 -specific propranolol and the β 2 -selective agonist terbutaline revealed comparable competition rates. This is proof of the β 2 -selective binding of Alexa-NA to the β 2 -AR (28,29).…”

“…Other specific ligands for the β 2 -AR have also been coupled with fluorescence markers in the past. Heithier et al labeled the β 1 /β 2 -specific antagonist CGP12177a with the dyes NBD, bodipy, and fluorescein and described the binding of those to cell preparations and intact cells with K D values in the picomolar range (29,30). In 1982, Henis et al synthesized NBD-labeled alprenolol (31), while the high level of nonspecific binding of this ligand to cell membrane preparations was described by Rademaker et al (32).…”

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

“…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).…”

Synthesis and Properties of Fluorescent .beta.-Adrenoceptor Ligands

“…Finally, the characteristics of CGP 12177 binding to recombinant human /32-adrenoceptors expressed in Sf9 cells are similar to those reported from human cell lines constitutively expressing ^-adrenoceptors (Boege et al, 1988;Heithier et al, 1988a;Staehelin et al, 1983). Comparing the Kn values obtained by displacement of radioactive [3H]CGP 12177 bound to /3-adrenoceptors of Sf9 cells by fluorescent BODIPY-CGP (Figure 2a) with those obtained by fluorometry of BODIPY-CGP bound to Sf9 cell membranes (Figure 2b) and with data obtained by direct photon counting of BODIPY-CGP bound to single intact cells (Figure 2c,d) shows that the favorable ligand binding properties of CGP 12177 are not compromised by the derivatization with BODIPY.…”

“…For example, both would be expected to react with a common sequencerecognizing antibody, but only the former would be expected to bind the ligand with specificity and high affinity (von Zastrow & Kobilka, 1992). Therefore, we have made efforts to synthesize specific and tight-binding fluorescent /3-adrenergic ligands (Heithier et al, 1988a;Henis et al, 1982). In previous work (Henis et al, 1982), NBD-alprenolol was used because CGP 12177 was not yet available.…”

Dynamics of β2-Adrenergic Receptor−Ligand Complexes on Living Cells

“…Competition experiments with the β 1 /β 2 -specific propranolol and the β 2 -selective agonist terbutaline revealed comparable competition rates. This is proof of the β 2 -selective binding of Alexa-NA to the β 2 -AR (28,29).…”

“…Other specific ligands for the β 2 -AR have also been coupled with fluorescence markers in the past. Heithier et al labeled the β 1 /β 2 -specific antagonist CGP12177a with the dyes NBD, bodipy, and fluorescein and described the binding of those to cell preparations and intact cells with K D values in the picomolar range (29,30). In 1982, Henis et al synthesized NBD-labeled alprenolol (31), while the high level of nonspecific binding of this ligand to cell membrane preparations was described by Rademaker et al (32).…”

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

“…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).…”

Synthesis and Properties of Fluorescent .beta.-Adrenoceptor Ligands

“…Finally, the characteristics of CGP 12177 binding to recombinant human /32-adrenoceptors expressed in Sf9 cells are similar to those reported from human cell lines constitutively expressing ^-adrenoceptors (Boege et al, 1988;Heithier et al, 1988a;Staehelin et al, 1983). Comparing the Kn values obtained by displacement of radioactive [3H]CGP 12177 bound to /3-adrenoceptors of Sf9 cells by fluorescent BODIPY-CGP (Figure 2a) with those obtained by fluorometry of BODIPY-CGP bound to Sf9 cell membranes (Figure 2b) and with data obtained by direct photon counting of BODIPY-CGP bound to single intact cells (Figure 2c,d) shows that the favorable ligand binding properties of CGP 12177 are not compromised by the derivatization with BODIPY.…”

“…For example, both would be expected to react with a common sequencerecognizing antibody, but only the former would be expected to bind the ligand with specificity and high affinity (von Zastrow & Kobilka, 1992). Therefore, we have made efforts to synthesize specific and tight-binding fluorescent /3-adrenergic ligands (Heithier et al, 1988a;Henis et al, 1982). In previous work (Henis et al, 1982), NBD-alprenolol was used because CGP 12177 was not yet available.…”

Dynamics of β2-Adrenergic Receptor−Ligand Complexes on Living Cells

“…Competition experiments with the β 1 /β 2 -specific propranolol and the β 2 -selective agonist terbutaline revealed comparable competition rates. This is proof of the β 2 -selective binding of Alexa-NA to the β 2 -AR (28,29).…”

“…Other specific ligands for the β 2 -AR have also been coupled with fluorescence markers in the past. Heithier et al labeled the β 1 /β 2 -specific antagonist CGP12177a with the dyes NBD, bodipy, and fluorescein and described the binding of those to cell preparations and intact cells with K D values in the picomolar range (29,30). In 1982, Henis et al synthesized NBD-labeled alprenolol (31), while the high level of nonspecific binding of this ligand to cell membrane preparations was described by Rademaker et al (32).…”

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

“…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).…”