Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

Abstract

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β(2)-adrenergic receptors (β(2)AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β(2)ARs are constitutively immobile. About 2/3 of the β(2)ARs moved with a diffusion constant of D(2) = 0.03 ± 0.001 μm(2)/s and about 17% were diffusing five-fold faster (D(3) = 0.15 ± 0.02 μm(2)/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ(1) = 77 ± 1 ms and τ(2) = 388 ± 11 ms. Agonistic stimulation of the β(2)AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β(2)AR mobility suggesting that different receptor dynamics characterize different receptor states.

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

“…Techniques like FCS helped to define structure-function relationships among plasma membrane receptors (Caballero-George et al, 2012;Owen et al, 2009;Sieben, Kaminski, Kubitscheck, & Häberlein, 2011;Suzuki, Ritchie, Kajikawa, Fujiwara, & Kusumi, 2005). For example, epinephrine was found to induce dynamic regulations of the β 2adrenergic receptor exhibiting very rapid lateral diffusion (288 × 10 −10 cm 2 /s) for several minutes before reducing its mobility to 10 × 10 −10 cm 2 /s.…”

A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the β2-adrenergic responsiveness of human airway smooth muscle cells

“…Sieben et al have been able to demonstrate that under stimulating conditions the number of ß 2 AR increases with hindered lateral mobility [16]. This finding indicates a possible internalization, since it can be assumed that ß 2 AR are nearly immobile within coated pits.…”

“…By means of high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis 17 ingredients were identified from an ivy leaves dry extract (EA 575, DER 5-7.5:1, 30% m/m EtOH), leading to the detection of protocatechuic acid (1), neochlorogenic acid (2), chlorogenic acid (3), rutin (4), kaempferol-3-O-rutinoside (5), 3,4-, 3,5-and 4,5-dicaffeoylquinic acid (6,7,8) as well as hederacoside B, C, D and F (9,10,11,12), hederaginin-3-O-glucoside (13), a-, b-and d-hederin (14,15,16). In addition, cryptochlorogenic acid (17) was identified in ivy leaves dry extracts for the first time.…”

Exogenous norepinephrine attenuates the efficacy of sunitinib in a mouse cancer model

“…Since the expression of β-ARs was observed in human lung adenocarcinoma A549 cells [65,66], only an immunohistochemical analysis for β-ARs in B16F1 cells was carried out. Hegener et al [65] also found that the internalization and endocytosis of β2-AR in A549 cells were stimulated by terbutaline (selective β2-AR agonist) and forskolin (cAMP analogs), whereas blocked by propranolol.…”