Novel tools for investigating GPCR-mediated 14-3-3 signaling pathway

DESCRIPTION provided by applicant It is now clear that GPCR signaling is pluridimensional Defining mechanisms for receptor signaling pathways is essential for understanding many aspects of cell biology as well as for effectively targeting signaling pathways for drug discovery Ligands of receptors do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others Signaling pathway specific ligands or biased ligands that selectively activate one pathway over another as well as behave as agonists in one pathway but antagonists in another pathway have greatly impacted our understanding of intricate GPCR signaling pathways and GPCR drug discovery In addition to G protein and arrestin signaling pathways signal adaptor protein is another cellular effectors activated by GPCRs The first evidence of as a cellular effector of GPCRs was demonstrated with the adrenergic receptors ARs Interaction of and ARs is ligand dependent proteins are ubiquitously expressed in cells but their highest expression is found in the brain proteins have been implicated in a number of neurological disorders such as Alzheimerandapos s disease Parkinsonandapos s disease schizophrenia bipolar disorder based on evidence from both clinical and laboratory studies Similar to arrestins proteins have no intrinsic enzymatic activity but bring two or more proteins together to facilitate signal transduction processes A very recent bioinformatic analysis predicts neurotransmitter GPCRs including GRMs have binding motifs Interestingly GRMs family C GPCRs do not recruit arrestins like many GPCRs do when activated Do they utilize for additional signaling in addition to G proteins Lack of a user friendly and scalable too for assessing GPCR mediated signaling could be a major reason for the signaling pathway unexploited We plan to apply LinkLight technology to develop GPCR mediated signaling pathway assays We plan to use ADRB as a model to demonstrate the assay feasibility The assay utilizes ADRB and interaction or ADRB signal complex formation as the signal readout Our preliminary data transient expression and stable expression experiments showed ADRB interaction or signal complex formation is agonist concentration dependent We also compared ADRB and ADRB arrestin LinkLIght assays in response to various agonists partial agonists and antagonists The preliminary data showed that the overall patterns of various ligand responses were similar but there were some differences in relative signal strength and potency EC Interestingly ADRB antagonists based on G protein signaling showed a partial activity in ADRB arrestin assay but had no activity in ADRB assay Our preliminary results showed that ligands are not created equal based on different signaling pathways Based on the observation it prompts us to profile existing drugs for their arrestin and signaling pathways Historically GPCR drug discoveries rely on G protein signaling pathways to assess compound activity such as many old antipsychotic drugs Their activity on arrestin and signaling i unknown Although having demonstrated therapeutic benefits these drugs also have serious side effects Despite huge efforts spent by the pharmaceutical industry the options for developing safer and more efficacious antipsychotic drugs remain elusive Could therapeutic and side effects are due to specific signaling pathways I plan to use D R an important antipsychotic drug target as a model to investigate a panel of D R ligands for their activity on arrestin and signaling It is now known that ligands do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others Ligands could have signaling in addition to G protein and arrestin signaling Biased ligands could have differential activities on these signaling pathways We plan to take the advantage of multiplex ability of the LinkLight technology to establish a dual signaling pathway assay for assessing arrestin and signaling simultaneously We plan tasks for the proposal Task Using ADRB as a model to assess GPCR mediated signaling and generating stable pLuc reporter cell lines Task Assess the general applicability of GPCR signaling assays with brain derived GPCRs Task Profile and compare D R mediated and arrestin signaling pathways with known D R ligands Task Develop GPCR and GPCR arrestin dual signaling pathway assays The assay cell lines tools developed in the proposal will be commercially available We also plan to use the tools for commercial compound screening and profiling services A CDA and MTA have signed with a major US research reagent company for potential business opportunity collaboration PUBLIC HEALTH RELEVANCE This research proposal is in response to Program Announcement PA Number PA reissuing FOA PA for andquot Novel Tools for Investigating Brain derived GPCRs in Mental Health Researchandquot G protein coupled receptors GPCRs are major drug targets for mental health Upon ligand binding multiple cellular effectors are recruited to receptors These effectors likely elicit specific signaling cascades Multiple cellular effectors offer the possibility of crosstalk fine tuning and specifically regulaing GPCR signaling at multiple levels Signal adaptor protein is one of such cellular effectors engaged with GPCRs proteins are ubiquitously expressed in cells but their highest expression is found in the brain proteins have been implicated in a number of neurological disorders such as Alzheimerandapos s disease Parkinsonandapos s disease Schizophrenia Bipolar disorder based on evidence from both clinical and laboratory studies Although bioinformatic analysis predicts neurotransmitter GPCRs have binding motifs GPCR mediated signaling is poorly understood in contrast to G protein and arrestin dependent signaling pathways Lack of a robust and scalable tool to assess GPCR mediated signaling impedes progress toward development of drugs targeting this signaling pathway This proposal is to develop novel tools for assessing GPCR mediated signaling Elucidation of GPCR mediated signaling and use of the assay tools for screening compounds that may selectively modulate different GPCR signaling pathways is of scientific and commercial interest