We plan to develop novel tools for assessing GPCR ligands modulating G-protein-dependent and ?-arrestin- dependent ERK signaling pathways. Many GPCRs display a phenomenon in which different ligands can differentially activate different signaling pathways via the same receptor. It has become clear that single signaling pathway approaches for identifying drug candidates are not adequately suited to detect the full repertoire of compounds that may have other signaling pathway activities beyond the target receptor. One has to examine multiple signaling pathways to link pathway activities to physiologic functions. ERK signaling cascades are important components of GPCR signaling pathways. Both G-protein and ? -arrestin mediated signaling pathways can lead to ERK activation. G-protein activation of ERKs results in the translocation of active ERK to the nucleus, where it can phosphorylate and activate various transcription factors. In contrast, when ERK is activated via an arrestin-dependent mechanism, active ERKs remain largely in the cytoplasm, where they can phosphorylate non-nuclear substrates. Subcellular locations of phosphorylated ERKs determine downstream signal transduction cascades. Current ERK activation assays are all antibody-based methods. The antibody-based assays measure the phosphorylation status of ERKs using cell lysates, but lack the ability to distinguish G-protein-dependent or arrestin-dependent ERK activation. Critical information on subcellular location and distribution of activated ERKs, which determine specific signal transduction cascades, is missing. In addition, some of antibody based assay methods are not HTS friendly. Currently, pathway- specific ERK activation assays with the HTS capability do not exist. We propose to develop these novel tools by applying newly developed cell-based protein-protein interaction LinkLight assay technology. The assays can measure specific subcellular ERK activation signaling pathways. ERK signaling pathways are implicated in LTP, memory, learning, mood stabilization etc. Therefore, developing these tools for identifying compounds modulating pathway-specific ERK activation should facilitate new GPCR drug discovery efforts for treating mental disorders. We propose four aims for the project.
Aim 1. Develop G-protein-dependent ERK2 activation pathway assay. We plan to utilize phosphorylated ERK2 interaction with imp7 as signal readout for assay development. We plan to validate the assay by using the m-opioid receptor (MOR) and its ligand morphine for nuclear location of the ERK2 activation pathway, since morphine is known to activate G-protein-dependent ERK pathway.
Aim 2. Develop arrestin-dependent ERK2 activation pathway assay. We plan to utilize phosphorylated ERK2 interaction with receptor-associated ? -arrestins as signal readout for the assay development. We plan to validate the assay by using the MOR and its ligand etorphine for arrestin-dependent cytoplasmic location of the ERK2 activation pathway, since etophine is known to activate arrestin-dependent ERK pathway.
Aim 3. Generate and characterize D2R ERK pathway-selective activation assay cells. Dopamine D2 receptors (D2R) include a long (D2L) and a short (D2S) isoforms. We plan to generate stable D2L G-protein- dependent and D2S arrestin-dependent ERK activation assay cells. It is known that D2S activates ERK signaling by an arrestin-dependent pathway and D2L activates ERK signaling by an arrestin-independent pathway. The cells will be used in aim 4 for assessing D2R compounds on ERK pathway activities and for assessing HTS feasibility.
Aim 4. Assessing D2R compounds on specific ERK pathway activities and assessing HTS feasibility by conducting a small pilot screening. Brain-derived dopamine D2 receptor-regulated behaviors have been associated to ERK activation. However, it is largely unknown D2R compounds on ERK activation especially specific ERK activation cascades. We plan to profile D2R compounds (listed in ref. 44) to look for their ERK pathway-selectivity preferences. Dr. Sam Kongsamut, our consultant for the project, having over 20 years experience in antipsychotic drug discovery programs in pharmaceutical industry will correlate the specific ERK pathway assay potencies with potencies of in vivo studies (animal models and clinic data). Pathway-selective compounds could have functional selectivity. We also plan to demonstrate assay feasibility for HTS. Dr. Wei Zheng's group in NCATS will conduct the pilot screenings using the LOPAC library. Based on the results of the aim 4, we will plan next phase research proposal (phase II) and collaborate with partners for developing new leads for treatment of mental health disorders. Once developed, these cells would be the unique products on the market. We plan to market specific GPCR ERK LinkLight assay cells, provide custom-services for compound profiling and screening, and license ERK pLuc reporter host cells with an annual fee. The end users can use these host cells to develop their interested GPCR ERK LinkLight assays.
This research proposal is in response to Program Announcement (PA) Number: PA-10-081, reissuing FOA PA-06-375 for Novel Tools for Investigating Brain-derived GPCRs in Mental Health Research. We plan to develop novel tools for assessing G-protein-dependent and ? -arrestin-dependent ERK signaling pathways. It has become clear in recent years that multiple signaling transduction pathways are employed upon GPCR activation. One of the major cellular effectors activated by GPCRs is extracellular signal-regulated kinase (ERK). ERKs have been implicated in modulating synaptic plasticity and playing important roles in learning, memory and long-term potentiation. Recent studies coming from NIMH show altered ERK pathway components in psychiatric disorders including major depressive disorder, bipolar disorder, and schizophrenia. Both G-protein and ? -arrestin mediated signaling pathways can lead to ERK activation. G-protein activation of ERKs results in the translocation of active ERK to the nucleus, where it can phosphorylate and activate various transcription factors. In contrast, when ERK is activated via an arrestin-dependent mechanism, active ERKs largely remain in the cytoplasm, where they can phosphorylate non-nuclear substrates. Determining specific ERK signaling pathways activated by GPCR ligands has become very important in deciphering cellular physiological responses. The proposed assays would be the first such kind of tools to address GPCR activation on distinct ERK cellular signal cascades and will facilitate GPCR drug discovery for treating mental disorders.