Guillermo A. Yudowski - US grants

[unreadable] DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCR) are integral plasma membrane proteins that transduce external signals into the cell. GPCRs mediate many important signaling functions in the central nervous system (CMS) and mediate the effects of many therapeutic and abused drugs. A basic mechanism by which these receptors are regulated is by ligand-induced endocytosis. This process reduces the number of activated receptors at the cell surface therefore regulating receptor signaling. While endocytosis is strongly regulated by physiological ligands and drugs, it is generally believed that recycling is a nonregulated process. New evidence from our laboratory and others are challenging this default hypothesis of GPCR recycling. We have developed a technique to investigate single insertion events at the cell surface by combining live total reflection microscopy (TIRF) with a pH sensitive extracelullar tag fused to the extracelullar terminus of GPCRs. Our preliminary results indicate that receptor recycling is a tightly regulated process involved in the homeostatic response to extracelullar stimuli. Furthermore, regulation of recycling is dependent on receptor activation, which ultimately decreases the frequency of recycling events reducing receptor number at the cell surface. I propose to extend and further develop this novel approach to examine: a) The characteristics of the vesicles mediating receptor recycling, b) Identify the machinery involved in the fusion process, and its possible regulation by receptor activity, c) Investigate if the regulation of receptor recycling is a general process for GPCRs, d) Define and investigate the functionality of newly inserted receptors and the possible existence of a molecular complex associated to reinserted receptors. This work will be perfomed using an experimentally advantageous heterologous cell model, and the results will be later examined in hippocamapal neurons. By investigating the regulation of receptor insertion to the cell surface, our work has the potential to provide fundamental new insight to key cellular events likely to be relevant to a wide variety of physiological and pathological processes. Obtaining the K99/ROO award will be an extraordinary opportunity to transition from my postdoctoral studies to an independent position in a university/research institution. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are one of the largest families of transmembrane receptors and a major target of current therapeutic drugs. At the signaling level, it has become clear that ligands acting on the same receptor can activate multiple and sometime opposing signaling cascades; a process defined as functional selectivity or biased agonism. One of the main effectors of functional selectivity are beta-arrestins, multifunction proteins recruited to activated receptors. However, how receptor activation translates into beta-arrestin signaling is not clearly defined. Our preliminary work combining state-of-the-art live cell imaging with molecular and biochemical techniques identifies ligand-specific dwell times, the time receptors are clustered into individual endocytic pits before endocytosis, as a mechanism by which receptors can control beta-arrestin mediated signaling. Our hypothesis suggests that ligands induce specific phosphorylations at the receptor level, eliciting specific endocytic dwell times during which beta-arrestins remain recruited and engaged in signaling. We propose to define a mechanism by which the Cannabinoid 1 Receptor (CB1R), one of the most abundant receptors in the CNS and target of cannabis, controls beta-arrestin signaling during endocytic dwell times. Our aims are: 1) Characterize ligand-specific dwell times of the CB1R to test our hypothesis that ligands can elicit specific dwell times that ar independent of their endocytic efficacy. 2) Define the mechanisms underlying ligand-specific dwell times of the CB1R. We will test the hypothesis that dwell times are controlled by ligand-specific phosphorylation profiles (bar-codes) of the receptor. Alternative mechanisms will be also investigated. 3) Determine if beta-arrestin signaling is the physiological target of ligand specific dwell times in heterologous systems and native tissue. Finally, we will test different manipulations to control arrestin signaling by altering CB1R dwell times.

PROJECT SUMMARY The classic model of G protein coupled receptor (GPCR) activation centers on ligand binding, G protein activation, and signal transduction via G protein-mediated signaling events. This paradigm has been called into question however, with the finding that some ligands ?including endogenous ligands and therapeutic agents? have a preference for beta-arrestin mediated pathways. However, the information flow from receptor activation to signaling cascades and the mechanism of beta-arrestin signaling are not well understood. This proposal will elucidate, at the molecular level, the fundamental mechanisms controlling ligand-specific induction of beta- arrestin signaling with two highly clinically relevant GPCRs, the cannabinoid 2 receptor (CB2R) and the mu opioid receptor (MOR). These human receptors bind the plant-derived cannabinoids and opioids leading to psychostimulant effects and reduction of pain. Precise control of receptor activation and signaling is critical to obtain only the desired therapeutic results, however, and not the undesired side effects such as tolerance, drug abuse and dependence. Substantial preliminary studies identified ligand-specific dwell times, i.e. the time receptors are clustered into clathrin coated pits with beta-arrestins before endocytosis, as a mechanism controlling beta-arrestin signaling. This trafficking event can be chemically and genetically modulated to selectively control beta-arrestin signaling, providing novel therapeutic strategies. This project will combine state-of-the-art live cell imaging technologies (total internal reflection fluorescence and spinning disk microscopies), and biochemical approaches to determine if ligand-specific dwell times are a general event controlling beta-arrestin signaling.  Multiple ligands for these receptors will be investigated in heterologous systems and in cells endogenously expressing the receptors. Preliminary results in primary cultures strongly support our hypothesis that long dwell times correlate with beta-arrestin signaling. The aims are to: (1) examine endocytosis of the CBR2 and MOR at the single endocytic pit level, and (2) define the impact on cellular mechanisms of CB2R and MOR mediated beta-arrestin signaling, including whether endocytic dwell times can modulate these pathways. Results will provide a physiological role for the previously described variability in endocytic dwell times. These findings may be extended to future drug discovery efforts, including for other GPCRs, to rationally design therapeutic agents with specific outcomes in areas intractable via current technology.