2015 — 2017 |
Lobingier, Braden |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
The Role of Endosomal Sorting in Regulating Opioid Receptor Function @ University of California, San Francisco
? DESCRIPTION (provided by applicant): The physiological effects of opiates, including drugs of abuse such as heroin or prescription opiate medications, are mediated by G protein-coupled receptors (GPCRs). Activation of opioid GPCRs results in feedback mechanisms in which receptor number and signaling activity is regulated through endocytosis and sorting at the endosome. The current model describes three sorting operations for opioid GPCRs at the endosome: degrade at the lysosome, reinsert at the cell surface ('recycling'), or hold at the endosome ('retention'). The importance of this process in regulation of opioid function is underscored by the observation that the mu-opioid receptor (MOR) and the delta-opioid receptor DOR are sorted in diametrically opposed manners at the endosome: MOR recycles while DOR degrades. While the mechanisms and proteins involved in recycling and degradation of opioid receptors are becoming well understood, the role of the endosomal retention remains puzzling. The central paradox of endosomal retention comes from experimental data linking it to the sorting of DOR (which degrades) but also MOR (which recycles). These data raise the question of when and how endosomal retention occurs for opioid receptors in context of the general trafficking outcomes already described. Testing these questions has proven difficult, as for the last decade the only known component of the retention pathway was the protein GASP1 (GPCR-associated sorting protein 1), and its connection with the receptor is challenging to manipulate. An exciting recent development has been the identification of a new protein, named Beclin2, which forms a complex with GASP1 and is necessary for GASP1 function. Beclin2 is more amenable to experimental manipulation, opening the opportunity to directly test the role of endosomal retention in the regulation of opioid receptors. The hypothesis of this proposal, supported by preliminary data, is that the endosomal retention of opioid receptors functions as the initial sorting step for both the recycling and degrading pathways: Beclin2- GASP1 hold opioid GPCRs at endosomes until a handoff of the receptor mediates spatial separation and entry into the recycling or degrading pathways. This hypothesis will be rigorously tested using a combination of Determine the role of Beclin2 in sorting of defined manipulations (to the receptors and endosomal sorting machinery) with techniques including flow cytometry, microscopy, biochemistry, and mass spectrometry in primary neuronal cultures and non-neuronal recycling GPCRs; (2) Define the relative functional locations of Beclin2 and the endosomal recycling model cell lines. The specific aims of this proposal: (1) complex; (3) Establish the identity of the Beclin2 sorting complex. This proposal addresses opioid receptor regulation on the molecular level with the goal of better understanding the cellular response to opiate drug abuse and addiction.
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0.915 |
2018 — 2021 |
Lobingier, Braden |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Molecular Basis For Ligand and Cell Type Specific Regulation of Opioid Receptors @ University of California, San Francisco
Abstract/Project Summary. Opioid abuse and addiction are major public health concerns. Opioids are highly useful analgesics, yet an estimated two million people in the United States suffer disorders related to abuse of prescription opioids. The social and economic costs of these disorders are devastating and on the rise: an average of 44 people die every day from prescription opioid overdoses. Opioids are structurally diverse molecules that include oxycodone, heroin and endorphins, and the physiological effects of these molecules are mediated by G protein-coupled receptors (GPCRs). Recently developed `biased' opioid agonists demonstrate that this diversity can be mined to identify drugs with less harmful side effects, but the mechanism of action of these `biased' agonists remains incompletely resolved. New technical advances now make it possible to biochemically capture the protein interaction networks mediating GPCR activity from inside of living cells with sub-minute temporal resolution. The ability to capture, quantify, and characterize the endogenous proteins which mediate and regulate opioid activity opens new doors for determining how biased agonists differ from endogenous ligands or drugs of abuse. This K99/R00 award combines critical new training in cutting-edge proteomics with traditional cell biological techniques to examine the mechanism of biased opioid agonism: Aim 1-Define the kinetics by which different classes of opioids alter mu opioid receptor (µOR) location and coupling to its transducer and regulatory proteins; Aim 2-Identify new protein regulators of µOR stimulated by standard or biased opioids. Aim 3- Define µOR signaling targets and determine if these proteins differ between opioids. Future studies based on these results will help to define how opioid receptors operate under normal, pharmacologically activated, or disease states resulting from drug abuse and addiction.
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0.915 |
2020 — 2021 |
Lobingier, Braden |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Endosomes as a Multifunctional Hub to Control Gpcr Function @ Oregon Health & Science University
Project Summary/Abstract GPCRs are the largest family of membrane bound signaling molecules and, collectively, the target of many drugs currently used in the clinic. Classically, GPCRs were thought to be active at the cell surface and inactive while undergoing molecular sorting and trafficking events within the cell. Recent work has overturned this model. GPCRs are not quiescent inside of cells. Instead, it is now known that GPCRs can activate G protein signaling from many intracellular compartments including the endosome, and this intracellular signaling changes drug response. While efforts are already underway to harness endosomal GPCR signaling as a drug target, much is unknown about GPCR sorting at endosomes and how these trafficking processes control endosomal GPCR signaling. The goal of this proposal is to address the knowledge gap surrounding GPCR sorting at endosomes, and to determine how these pathways control endosomal signaling. In Project 1 we test the hypothesis that endosomal sorting functions as a kinetic timer to control GPCR signaling at endosomes. We examine a prototypical GPCR, the beta 2 adrenergic receptor, and the use a combination of genetic engineering and proteomics to determine how sorting controls endosomal signaling. In Project 2 we focus on two different GPCRs which signal at endosomes but lack any of the consensus endosomal sorting motifs. We use a combination of chemical biology, genomics, and proteomics to identify the proteins and pathways which mediate endosomal sorting of these receptors. Our studies seek to reveal fundamental lessons about conserved cell biological pathways while driving forward a new area for future GPCR drug development.
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0.915 |