Detection of the secondary, low‐affinity β 1 ‐adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY‐TMR ‐ CGP

Background and Purpose CGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β 1 ‐adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low‐affinity β 1 ‐adrenoceptor site or conformation. β‐blocker affinities for this ‘ CGP 12177’ site of the human β 1 ‐adrenoceptor have thus far only been characterized in functional studies. Here, we used the fluorescent CGP 12177 analogue BODIPY‐TMR ‐ CGP to directly investigate receptor–ligand interactions at the secondary binding site of the β 1 ‐adrenoceptor. Experimental Approach The human β 1 ‐adrenoceptor was stably expressed in CHO cells containing a cAMP response element ( CRE ) ‐ secreted placental alkaline phosphatase ( SPAP ) reporter gene construct. Functional responses of BODIPY‐TMR ‐ CGP were determined in the CRE‐SPAP reporter gene assay, and manual and automated confocal microscopy platforms used to investigate the binding properties of BODIPY‐TMR ‐ CGP . Key Results BODIPY‐TMR ‐ CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β 1 ‐adrenoceptor site. In confocal microscopy studies, specific BODIPY‐TMR ‐ CGP binding allowed clear visualization of β 1 ‐adrenoceptors in live cells. Using a wider concentration range of labelled ligand in a high‐content fluorescence‐based binding assay than is possible in radioligand binding assays, two‐site inhibition binding curves of β‐adrenoceptor antagonists were revealed in CHO cells expressing the human β 1 ‐adrenoceptor, but not the β 2 ‐adrenoceptor. Conclusions and Implications The fluorescent CGP 12177 analogue allowed the detection of the β 1 ‐adrenoceptor secondary site in both functional and binding studies. This suggests that BODIPY‐TMR ‐ CGP presents an important and novel fluorescent tool to investigate the nature of the secondary β 1 ‐adrenoceptor site.