Dynamics of beta2-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 K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-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 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.
Hegener
Oliver
O
Department of Pharmaceutical Biology, Philipps University, Deutschhausstrasse 17A, 35032 Marburg, Germany.
Prenner
Lars
L
Runkel
Frank
F
Baader
Stephan Leonhardt
SL
Kappler
Joachim
J
Häberlein
Hanns
H
D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D015394:Molecular Structure; D009474:Neurons / Q000378:metabolism*; D009638:Norepinephrine / Q000031:analogs & derivatives; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D012503:Saponins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013726:Terbutaline / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D005456:Fluorescent Dyes; D008024:Ligands; D020033:Protein Isoforms; D018343:Receptors, Adrenergic, beta-2; D012503:Saponins; C010177:beta-hederin; D005576:Colforsin; D009828:Oleanolic Acid; D013726:Terbutaline; D009638:Norepinephrine