Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of -9.53 and -8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]-butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol. 1983, 5, 430-437.

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

PHASE SEPARATION AND DOMAIN INTERACTION IN PHEOPHYTIN CONTAINING MONOLAYERS

On a mesuré simultanément la pression de surface et l'intensité de fluorescence des couches minces monomoléculaires qui se composent du lipide contenant de la phéophytine à la surface de séparation de l'eau et de l'air. Nous avons trouvé une séparation de deux phases différentes. La phase 1 en domaines de phéophytine, par contre la phase 2 est composée d'un seul domaine de lipide contenant 2 à 15 % de phéophytine dépendant de l'état du lipide. La transition des domaines phéophytiniques est décrite par un modèle qui explique correctement la dépendance observée de la pression de transition πk de la concentration. L'analyse des résultats prouve que les domaines phéophytiniques sont étendus à deux dimensions et qu'ils sont de grandeur limitée (plus petites que 1 000 molécules). Les domaines sont stabilisés par des forces stériques provenant des environs. Nous avons calculé le changement des interactions pendant la transition de phase et nous avons obtenu le résultat suivant : 0,6 x 10-13 erg par molécule au milieu et 1,2 x 10-13 erg par molécule au bord du domaine.Surface pressure and fluorescence intensity of pheophytin containing lipid-monolayers are measured simultaneously at the air-water interface. A phase separation into two distinct phases is reported. Phase 1 exists of pheophytin domains whereas phase 2 is a lipid domain containing pheophytin in molar content between 2 % and 15 %, depending on the state of the lipid. The transition of the pheophytin domains is described within a model explaining correctly the observed concentration dependence of the transition pressure πk. The analysis shows that the pheophytin domains are two-dimensional and of limited size (smaller than 1 000 molecules). The domains are stabilized by their environment by steric forces. The change of the interactions during the phase transition is calculated. It amounts to 0.6 x 10-13 erg per molecule in the bulk of the domain and to 1.2 x 10-13 erg per molecule at the domain wall

Fluorescein-Labelled Glucagon: A New Probe for the Study of Receptor Disposition in Membranes

New fluorescent glucagon derivatives were synthesized by converting tryptophan25 to 2-thiol-tryptophan with the consequent use of thiol specific fluorescent reagents. All derivatives retained the ability to bind tightly to rat liver membranes and rat hepatocytes in primary culture and to activate adenylate cyclase as potently as native glucagon. Thus these derivatives are full agonists. From experiments with monolayer cultured hepatocytes and 125I-glucagon at elevated temperatures it was assumed that the ligand was internalised at this temperature since some of the specifically bound ligand could no longer be washed off with acid. This was confirmed in experiments where monolayer cultures of hepatocytes were incubated with the fluorescein-labelled derivates of glucagon, thus allowing the study of the distribution of glucagon specifically bound on the cell surface using video intensification microscopic techniques. In keeping with autoradiographic studies using radiolabelled glucagon, or electron microscope studies using ferritin labelled glucagon, we could now show using fluorescently labelled glucagon derivatives and video intensification microscopy that at lower temperatures the bound ligand was distributed all over the cell surface. Whereas, at the higher temperature, ligand derived fluorescence could only be detected in mobile intracellular vesicles following internalisation and removal from the cell surface

Synthesis and properties of fluorescent ß-adrenoceptor ligands

We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human ß2-adrenoceptor gene and expressing (2-3.5) x 105 human ß2-adrenoceptors per cell. Among these derivatives only BODIPY-CGP gave a receptor-specific signal sufficiently strong for measuring the on- and off-rate constants and the equilibrium dissociation constant of beta-adrenoceptor-specific binding by spectrofluorometry or photon counting. Similar KD values for BODIPY-CGP binding were obtained by kinetic measurements (approx. 250 pM) and under equilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [3H]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 12177 to near background levels. The disposition of the ß2-adrenoceptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the cell surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenoceptors on a single cell can be assessed by photon counting under the fluorescence microscope. Cells can also be scanned by fluorescence-activated flow cytometry