Lateral motion of beta receptors in membranes of cultured liver cells.

Abstract

We have studied the lateral mobility and distribution of fi receptors on Chang human liver cells by fluorescence photobleaching recovery and video intensification microscopy.The ,3 receptors were labeled with the fluorescent antagonist 7-(2-allylphenoxy)-, 2 -dimethyl-6 -hydroxy-1 -(4 -nitrobenzo -2 -oxa-1,3-diazolyl)-1,4-diazaheptane (Alp-NBD). Sixty to 75% of the staining was specific (displaceable by unlabeled antagonists). Most of the antagonist-occupied I8 receptors were immobile, because only 15-25% of their fluorescence recovered on the experimental time scale at 23TC. This immobility correlates with the clustered distribution of Alp-NB)-/-receptor complexes at 40C and 37C. The .8 receptors appear to be aggregated prior to antagonist binding, because visible patches were observed immediately after labeling for 30 sec at 4C. Preincubation at 37°C with (-)-isoproterenol, a .8 agonist, prior to Alp-NBD labeling induced a time-dependent release of the /3 receptors to a more homogeneous distribution and increased the mobile fraction to 70-80% (lateral diffusion coefficient = 1.4 X 10-9 cm2/sec at 23°C). This is not due to an effect on membrane fluidity, because the diffusion coefficient of a lipid probe was not altered. The time course of agonist-induced fl-receptor mobilization correlates with receptor loss and adenylate cyclase desensitization but is much slower than adenylate cyclase activation. This indicates that adenylate cyclase activation by (3 receptors does not require macroscopic lateral mobility of the majority of the (3 receptors.Recent models of adenylate cyclase activation by /8 receptors stress the role of diffusional encounters between hormonereceptor complexes in the plasma membrane and other components of the cyclase system (1). The hybridization experiments of Schramm et al. (2) and the kinetic analyses of Levitzki et aL (3) are usually cited to support these models based on the floating receptor hypothesis (4). Lateral mobility of hormone-receptor complexes relative to the other cyclase components is supposed to be necessary for their effective interaction, although the receptor complexes themselves need not be mobile if the other components are. Recent studies of reconstituted systems (5) have, however, shown that the encounter of the hormonereceptor complex with the guanyl nucleotide binding protein (G protein) (6, 7) is much faster than the rate ofcyclase activation and so cannot be rate-limiting in the activation process. This result must still be tested in native membranes. Further consideration of the kinetic role of receptor-G protein encounters requires direct measurement of the lateral mobility and distribution of both the receptors and the G proteins in living cells. Here we report measurements of the distribution and lateral diffusion coefficients of / receptors labeled with a fluorescent antagonist on Chang human liver cells, in which the influence of membrane structure on ,B-adrenergic stimulation of adenylate cyclase has previously been studied (8). The experiments u...

Molecular probes for extracellular adenosine receptors

“…Fluorescent analogs of receptor ligands have a variety of applications [23,24], For example, spectral changes of fluorescent analogs have been used to characterize receptor properties in conformations associated with different functional states [12], accessibility of sites by fluorescence quenching, fluorescence depolarization, and energy transfer, and the distribution and lateral mobility of receptors in membranes using fluorescence photobleaching of NBD derivatives [11]. The direct visualization of receptors for thyrotropin-releasing hormone on a tumor cell line using fluorescent drug analogs has been reported [10].…”

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…Photobleaching studies have been used for decades to assess the mobility of GPCRs in the cell membrane (Henis et al, 1982;Dorsch et al, 2009), and these have been more recently complemented by single molecule observations of fluorescently bound ligands (Hern et al, 2010;Kasai et al, 2011) or receptors themselves . From these data it is evident that GPCRs represent moving targets, although there is considerable variation in the reported fraction of mobile versus immobile receptors.…”

Lateral mobility of proteins and lipids in the red cell membrane and the activation of adenylate cyclase by β‐adrenergic receptors

“…The few direct measurements of padrenergic receptor lateral mobility [6,7] suggested, for instance, that the receptor is immobile.…”