title: Detection of the secondary, low-affinity β1 -adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP.
abstract: OBJECTIVE
  CGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β1 -adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity β1 -adrenoceptor site or conformation. β-blocker affinities for this 'CGP 12177' site of the human β1 -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 β1 -adrenoceptor.
  
  
  METHODS
  The human β1 -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.
  
  
  RESULTS
  BODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β1 -adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of β1 -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 β1 -adrenoceptor, but not the β2 -adrenoceptor.
  
  
  CONCLUSIONS
  The fluorescent CGP 12177 analogue allowed the detection of the β1 -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 β1 -adrenoceptor site.
authors: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.
authors: K
authors: K
authors: Gherbi
authors: S J
authors: SJ
authors: Briddon
authors: S J
authors: SJ
authors: Hill
mesh_terms: D058665:Adrenergic beta-1 Receptor Agonists / Q000494:pharmacology*; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology*; D016466:CHO Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000235:genetics; D003517:Cyclopentanes / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D011412:Propanolamines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology*; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism*; D020218:Response Elements
chemical_list: D058665:Adrenergic beta-1 Receptor Agonists; C481870:BODIPY-TMR-CGP; D001896:Boron Compounds; D003517:Cyclopentanes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C509768:PDZK1IP1 protein, human; D011412:Propanolamines; D011758:Pyrroles; D018342:Receptors, Adrenergic, beta-1; D000242:Cyclic AMP; C033394:CGP 12177