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

Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells

<p>Abstract</p> <p>Background</p> <p>Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown.</p> <p>Results</p> <p>Here, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR₁) and 2 (PAR₂) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR₁and PAR₂agonists, but neither PAR₃nor PAR₄agonists, trigger dose-dependent Ca²⁺mobilization as well as G_q-type DMR signals. Both Ca²⁺flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR₁and PAR₂exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca²⁺flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities.</p> <p>Conclusion</p> <p>Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR₁and PAR₂in A431.</p

Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells-8

<p><b>Copyright information:</b></p><p>Taken from "Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells"</p><p>http://www.biomedcentral.com/1471-2121/8/24</p><p>BMC Cell Biology 2007;8():24-24.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1925066.</p><p></p>ypsin (100 nM), and thrombin (40 unit/ml). The cell responses with the pre-treatment with the HBSS only were also included as control

Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells-0

<p><b>Copyright information:</b></p><p>Taken from "Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells"</p><p>http://www.biomedcentral.com/1471-2121/8/24</p><p>BMC Cell Biology 2007;8():24-24.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1925066.</p><p></p>Gsignaling, which proceeds through activation of the receptor, its coupled G protein and downstream target phospholipase C (PLC). The PLC hydrolyzes the membrane lipid phosphatidylinositol bisphosphate (PIP), producing inositol triphosphate (IP) and diacylglycerol (DAG). IPbinds to and opens a calcium channel in the endoplasmic reticulum, leading to calcium mobilization. Calcium alters many cellular processes. The interaction of both DAG and calcium with protein kinase C (PKC) activates PKC kinase activity, which, in turn, phosphorylates many different protein targets including small GTPase Rho, leading to the remodeling of cytoskeletal structure. (b) The increase in intracellular Calevel as a function of the concentration of different soluble PAR agonists. (c) The real-time dynamic mass redistribution signals induced by SFLLR-amide at different doses. The solid arrow indicates the time when SFLLR-amide is introduced. The DMR consists of two phases: an increase signal (termed Positive-DMR, P-DMR) and a sequential decay signal (termed Negative-DMR, N-DMR). (d) The amplitudes of both P-DMR and N-DMR events, calculated as indicated in (c), as a function of SFLLR-amide concentration

Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells-10

<p><b>Copyright information:</b></p><p>Taken from "Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells"</p><p>http://www.biomedcentral.com/1471-2121/8/24</p><p>BMC Cell Biology 2007;8():24-24.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1925066.</p><p></p>GRL-amide (20 μM), SLIGKV-amide (20 μM), thrombin (40 unit/ml), SFLLR-amide (20 μM), trypsin (1024 nM), SFLLR-amide+SLIGRL-amide (each at 20 μM). (b) Comparison of the maximal DMR and Camobilization responses induced by different PAR agonists. The DMR response was calculated using the amplitude of the P-DMR event. Since trypsin at doses greater than ~1000 nM led to significant cell detachment (ref. 16), the DMR signal induced by trypsin at 1024 nM was used as its maximal response

Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells-2

<p><b>Copyright information:</b></p><p>Taken from "Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells"</p><p>http://www.biomedcentral.com/1471-2121/8/24</p><p>BMC Cell Biology 2007;8():24-24.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1925066.</p><p></p>of the P-DMR events induced by each agonist (40 unit/ml thrombin, 20 μM SFLLR-amide, or 20 μM SLIGKV-amide) were plotted as a function of YFLLRNP-amide concentration

Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells-4

<p><b>Copyright information:</b></p><p>Taken from "Optical biosensor differentiates signaling of endogenous PARand PARin A431 cells"</p><p>http://www.biomedcentral.com/1471-2121/8/24</p><p>BMC Cell Biology 2007;8():24-24.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1925066.</p><p></p>combinations of agonists. The agonist concentration was 40 unit/ml, 200 nM, 20 μM, 20 μM, and 100 nM for thrombin, trypsin, SFLLR-amide, SLIGKV-amide, and bradykinin, respectively