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 'default1 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 indicates that receptor recycling is a tightly regulated process involved in the homeostatic response to extracelullar stimuli. Furtheremore, 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 latter 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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Transition Award (R00)
Project #
5R00DA023444-05
Application #
8432875
Study Section
Special Emphasis Panel (NSS)
Program Officer
Pilotte, Nancy S
Project Start
2011-03-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2015-02-28
Support Year
5
Fiscal Year
2013
Total Cost
$229,758
Indirect Cost
$76,586
Name
University of Puerto Rico Med Sciences
Department
Type
Schools of Medicine
DUNS #
948108063
City
San Juan
State
PR
Country
United States
Zip Code
00936
Delgado-Peraza, Francheska; Ahn, Kwang H; Nogueras-Ortiz, Carlos et al. (2016) Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor. Mol Pharmacol 89:618-29
Palacio, Stephanie; Velázquez-Marrero, Cristina; Marrero, Héctor G et al. (2015) Time-Dependent Effects of Ethanol on BK Channel Expression and Trafficking in Hippocampal Neurons. Alcohol Clin Exp Res 39:1619-31
Roman-Vendrell, Cristina; Yudowski, Guillermo Ariel (2015) Real-time imaging of mu opioid receptors by total internal reflection fluorescence microscopy. Methods Mol Biol 1230:79-86
Roman-Vendrell, Cristina; Chevalier, Michael; Acevedo-Canabal, Agnes M et al. (2014) Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures. Front Cell Neurosci 8:363
Flores-Otero, Jacqueline; Ahn, Kwang H; Delgado-Peraza, Francheska et al. (2014) Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1. Nat Commun 5:4589
Montiel-Gonzalez, Maria Fernanda; Vallecillo-Viejo, Isabel; Yudowski, Guillermo A et al. (2013) Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc Natl Acad Sci U S A 110:18285-90
Yudowski, Guillermo A; Olsen, Olav; Adesnik, Hillel et al. (2013) Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses. PLoS One 8:e53965
Roman-Vendrell, Cristina; Yu, Y Joy; Yudowski, Guillermo Ariel (2012) Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists. J Biol Chem 287:14782-91
Yudowski, Guillermo A; von Zastrow, Mark (2011) Investigating G protein-coupled receptor endocytosis and trafficking by TIR-FM. Methods Mol Biol 756:325-32
Yu, Y Joy; Dhavan, Rani; Chevalier, Michael W et al. (2010) Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion. J Neurosci 30:11703-14

Showing the most recent 10 out of 11 publications