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

The agonist-induced dynamic regulation of the β2-adrenergic receptor (β2-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the β2-AR of neurons with a K D value of 1.29 ± 0.31 nM and of A549 cells with a K D of 5.98 ± 1.62 nM. The receptor density equaled 4.5 ± 0.9 μm-2 in neurons (ρN) and 19.9 ± 2.0 μm-2 in A549 cells (ρA549). Kinetic experiments revealed comparable on-rate constants in both cell types (k on = 0.49 ± 0.03 s-1 nM-1 in neurons and k on = 0.12 ± 0.02 s-1 nM-1 in A549 cells). In addition to the free ligand diffusing with a D free of (2.11 ± 0.04) × 10-6 cm2/s, in both cell types receptor−ligand complexes with two distinct diffusion coefficients, D bound1 (fast lateral mobility) and D bound2 (hindered mobility), were observed [D bound1 = (5.23 ± 0.64) × 10-8 cm2/s and D bound2 = (6.05 ± 0.23) × 10-10 cm2/s for neurons, and D bound1 = (2.88 ± 1.72) × 10-8 cm2/s and D bound2 = (1.01 ± 0.46) × 10-9 cm2/s for A549 cells]. Fast lateral mobility of the receptor−ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D bound2) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15−20 min in A549 cells (up to 40% of total binding). Thus, the receptor−ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor−ligand complexes toward D bound2. Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor−ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 μM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin α-hederin.