Agonist-Directed Desensitization of the β2-Adrenergic Receptor

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level

Receptor internalization and ERK1/2 phosphorylation are dependent on the agonist exposure time.

<p>(a)–(d) Confocal images of cells after immunostained with anti-β₂AR. The cells were first stimulated with 2 nM epinephrine for 1 min (a), 2 min (b), 5 min (c) and 30 min (d). After epinephrine removal, the cells were further incubated in medium at 37°C for 29 min (a), 28 min (b), 25 min (c) and 0 min (d), respectively, such that equal time post stimulation was achieved for all. The staining was performed using anti-β₂AR antibody and Alexa Fluor 488 goat anti-rabbit antibody. The nuclei were stained with DAPI. Confocal microscopy images were acquired on Zeiss confocal laser scanning microscope (oil-immersion, 63× objective). The green color is from the β₂AR, the blue from the nuclei. (e) The agonist exposure time and Ro31-8220 sensitivity of pERK stimulated by epinephrine in A431 cells. The cells were incubated with the vehicle (0 min) or 2 nM epinephrine for a specific time (1 min, 2 min, 30 min), followed by further incubation at 37°C for another time to ensure all reach 30 min post simulation. Ro31-8220 when used was presented throughout the incubation. Equal amounts of cell lysate were separated by SDS-PAGE and analyzed for pERK by Western blotting. Equivalent gel loading was confirmed by probing with antibodies against GAPDH.</p

The desensitization and resensitization patterns of quiescent A431 cells induced by epinephrine is sensitive to stimulation duration and several inhibitors.

<p>(a) The DMR signals upon repeated stimulations with epinephrine. After initial baseline (∼2 min), the cells were first stimulated with epinephrine (1^st EPI) for three different durations (1 min, 2 min, or 5 min), followed by perfusion with the assay vehicle for ∼35 min, and finally stimulated again with a continuous flow of epinephrine (2^nd EPI) for ∼30 min. (b) The amplitudes of both N-DMR and P-DMR of the 2^nd EPI induced DMR as a function of the 1^st stimulation conditions. (c) The epinephrine DMR in the absence and presence of dynasore. The cells were first stimulated with epinephrine for 2 min (1^st EPI) in the presence and absence of dynasore, followed by perfusion with the assay buffer in the absence and presence of dynasore, respectively. Afterwards, all the cells were repeatedly stimulated with epinephrine in the absence of dynasore (2^nd EPI). (d) The amplitudes of both N-DMR and P-DMR of the 2^nd EPI induced DMR as a function of inhibitors. Each inhibitor or their combinations were assayed in a manner similar to dynasore. The flow rate was 1 µl/min under all conditions. Inhibitor concentrations were 5 µM, 10 µM, 10 µM, and 50 µM for H-89, Ro31-8220, PP1, and dynasore, respectively. Epinephrine dose was 2 nM for all. n = 3.</p

The simulated dose responses of distinct agonists as a function of spare receptors in the cell using the operational model.

<p>(a) epinephrine; (b) pindolol; (c) salbutamol; and (d) salmeterol. The spare receptors were normalized in percentage.</p

The simulated sensitivity of distinct agonists to loss of receptors (in percentage).

<p>(a) The effect of an EC₁₀₀ of each agonist (calculated where the total receptor is unchanged; <i>R_T</i> = 100) at different receptor numbers. Data were normalized to percentage of the response at the EC₁₀₀ where <i>R_T</i> = 100. (d) The function of <i>E_max</i> achievable by the agonists as receptor loss.</p

The DMR patterns of cells upon repeated stimulation with pindolol and epinephrine.

<p>Step 1: 2 min pulse stimulation in the absence and presence of an inhibitor, followed by perfusion with the assay buffer in the absence and presence of the respective inhibitor; and Step 2: continuous exposure to 2 nM epinephrine using perfusion. (a) DMR signals, and (b) DMR characteristics, later of which the P-DMR of the 1^st pindolol-induced DMR, and both N-DMR and P-DMR of the 2^nd EPI induced DMR were plotted as a function of inhibitors. The flow rate was 1 µl/min under all conditions. n = 2 to 4.</p