Dynamics of β2-adrenergic receptor -: Ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor beta(2)-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 beta(2)-AR of neurons with a K-D value of 1.29 +/- 0.31 nM and of A549 cells with a KD of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 mum(-2) in neurons (rho(N)) and 19.9 +/- 2.0 mum(-2) in A549 cells (rho(A549)). 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) x 10(-6) cm(2)/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) x 10(-8) cm(2)/s and D-bound2 = (6.05 +/- 0.23) x 10(-10) cm(2)/S for neurons, and D-bound1 = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D-bound2 = (1.01 +/- 0.46) x 10(-9) cm(2)/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 muM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.