The spatiotemporal regulation of intracellular trafficking and signaling of G protein-coupled receptors (GPCRs) is a critical aspect of integrated responses of the cell to hormones. Indeed, defective transport and dysfunction of GPCRs are associated with the pathogenesis of many human diseases. Our overall objective is to define the molecular mechanisms underlying the maturation and signal propagation of GPCRs and their roles in modulating cellular responses to hormones and drugs. Under this broad objective, the focus of the current proposal is to elucidate the mechanisms of nascent GPCR export from the endoplasmic reticulum (ER) to the cell surface and GPCR-mediated activation of the mitogen-activated protein kinase pathway in neuroblastoma-glioma NG108 and human embryonic kidney HEK293 cell lines by using alpha2B-adrenergic receptor (alpha2B-AR) as a model GPCR. We have demonstrated that alpha2B-AR export from the ER is modulated by a highly conserved triple arginine (3R) motif. The 3R motif mediates receptor interaction with selective Sec24 isoforms, components of COPII-coated transport vesicles. Our studies have also revealed a novel function for GGAs [monomeric Golgi-localizing, 3-adaptin ear domain homology, ADP ribosylation factor (ARF)-binding proteins]. GGAs associate with alpha2B-AR and are required for alpha2B-AR transport from the trans-Golgi network (TGN) to the plasma membrane. Furthermore, we have identified a novel signaling pathway in which the di-tryptophan motif-mediated, agonist-dependent interaction of alpha2B-AR with the small GTPase ARF1 dictates the activation of the conventional Raf1-MEK-ERK1/2 cascade by the receptor.
The Specific Aims are: 1) to elucidate the mechanism of COPII vesicle-mediated alpha2B-AR export from the ER, 2) to determine the function of GGAs in alpha2B-AR transport from the TGN to the cell surface, and 3) to define the function and mechanism of alpha2B-AR- and ARF1-mediated activation of the Raf1-MEK-ERK1/2 pathway. Overall, these studies will reveal novel molecular mechanisms underlying export trafficking and signal regulation of GPCRs. The information generated from these studies may open new directions for designing drugs to treat diseases involving abnormal trafficking and functioning of GPCRs.

Public Health Relevance

This proposal will study the intracellular trafficking and functional regulation of G protein-coupled receptors. These receptors regulate a variety of cell functions under physiological and pathological conditions and are the targets for drugs to treat many diseases. The successful completion of these studies will open a new direction for designing drugs to treat human diseases involving abnormal trafficking and functioning of G protein-coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076167-08
Application #
8459462
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Ainsztein, Alexandra M
Project Start
2006-05-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
8
Fiscal Year
2013
Total Cost
$292,106
Indirect Cost
$97,369
Name
Georgia Regents University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Bertuccio, Claudia A; Lee, Shih-Liang; Wu, Guangyu et al. (2014) Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent. PLoS One 9:e92013
Zhang, Xiaoping; Wang, Hong; Duvernay, Matthew T et al. (2013) The angiotensin II type 1 receptor C-terminal Lys residues interact with tubulin and modulate receptor export trafficking. PLoS One 8:e57805
Wu, Guangyu (2013) Identification of endoplasmic reticulum export motifs for G protein-coupled receptors. Methods Enzymol 521:189-202
Dong, Chunmin; Wu, Guangyu (2013) G-protein-coupled receptor interaction with small GTPases. Methods Enzymol 522:97-108
Wang, Guansong; Wu, Guangyu (2012) Small GTPase regulation of GPCR anterograde trafficking. Trends Pharmacol Sci 33:28-34
Dong, Chunmin; Nichols, Charles D; Guo, Jianhui et al. (2012) A triple arg motif mediates ?(2B)-adrenergic receptor interaction with Sec24C/D and export. Traffic 13:857-68
Dong, Chunmin; Li, Chunman; Wu, Guangyu (2011) Regulation of ýý(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1. J Biol Chem 286:43361-9
Zhang, Xiaoping; Dong, Chunmin; Wu, Qiong J et al. (2011) Di-acidic motifs in the membrane-distal C termini modulate the transport of angiotensin II receptors from the endoplasmic reticulum to the cell surface. J Biol Chem 286:20525-35
Duvernay, Matthew T; Wang, Hong; Dong, Chunmin et al. (2011) Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface. J Biol Chem 286:14080-9
Qin, Kou; Dong, Chunmin; Wu, Guangyu et al. (2011) Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers. Nat Chem Biol 7:740-7

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