Document Detail


Dynamics of beta2-adrenergic receptor-ligand complexes on living cells.
MedLine Citation:
PMID:  15147203     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Oliver Hegener; Lars Prenner; Frank Runkel; Stephan Leonhardt Baader; Joachim Kappler; Hanns Häberlein
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  43     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2004 May 
Date Detail:
Created Date:  2004-05-18     Completed Date:  2004-08-23     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6190-9     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Biology, Philipps University, Deutschhausstrasse 17A, 35032 Marburg, Germany.
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MeSH Terms
Descriptor/Qualifier:
Adrenergic beta-Agonists / metabolism
Animals
Cell Line
Endocytosis / physiology
Epithelial Cells / cytology,  metabolism
Fluorescent Dyes / chemistry,  metabolism
Forskolin / metabolism
Hippocampus / cytology
Humans
Immunohistochemistry
Ligands
Molecular Structure
Neurons / metabolism*
Norepinephrine / analogs & derivatives
Oleanolic Acid / analogs & derivatives*,  chemistry,  metabolism
Protein Isoforms / metabolism
Pulmonary Alveoli / cytology
Rats
Rats, Sprague-Dawley
Receptors, Adrenergic, beta-2 / metabolism*
Saponins / chemistry,  metabolism
Spectrometry, Fluorescence / methods
Terbutaline / metabolism
Chemical
Reg. No./Substance:
0/Adrenergic beta-Agonists; 0/Fluorescent Dyes; 0/Ligands; 0/Protein Isoforms; 0/Receptors, Adrenergic, beta-2; 0/Saponins; 23031-25-6/Terbutaline; 27013-91-8/alpha-hederin; 508-02-1/Oleanolic Acid; 51-41-2/Norepinephrine; 66428-89-5/Forskolin

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