Heterotrimeric (a??) G proteins mediate signals from G protein coupled receptors (GPCRs), which play roles in a vast range of biological processes important for health and disease, and are the most common pharmaceutical drug targets. Although GPCRs are fairly common drug targets for neurological and cardiovascular diseases, there are fewer examples in the field of inflammation despite the fact that GPCRs can modulate T cell signaling. This proposal seeks to determine whether G protein ?? subunits might be useful drug targets in this area. Silencing the G protein ?1 subunit using small interfering RNA (siRNA) or inhibition of ?? signaling with the small molecule, 1 gallein, leads to increased T cell receptor (TCR)-stimulated production of the cytokine, interleukin 2 (IL-2) in CD4+ T cells. Potentially this effect could be used to enhance inflammation for vaccine development, tumor- immunotherapy, and also for treating systemic lupus erythematosis (SLE). The overall goal of this proposal is to elucidate the mechanisms by which ?? complexes modulate T cell interleukin 2 (IL-2) production and to assess their utility as novel targets for treating aberrant T cell signaling. The proposed experiments will test the following hypotheses in primary human T cells and/or the Jurkat T leukemia cell line. 1. G protein ?1? complexes inhibit TCR-dependent IL-2 production. To elucidate the mechanism of this effect, two hypotheses will be tested. (A) ?1? complexes may inhibit TCR-dependent IL-2 production by decreasing levels of diacylglycerol (DAG) and Ras/ MAPK activation. (B) Silencing of specific 23 complexes may be required for potentiation of TCR-stimulated IL-2. 2. as?1? stimulated by the A2AR, but not EP4R, inhibits TCR-dependent IL-2 production. The following possible mechanisms for this difference will be tested. (A) The two GPCRs may differentially affect DAG metabolism and Ras/MAPK signaling. (B) The spatial and/or temporal characteristics of cAMP responses to the two GPCRs may differ. (C) Different as?? complex(es) may be activated by the two GPCRs and exert different effects as a result s of their GPCR-stimulated activation, dissociation, and/or internalization patterns. 3. Targeting A2AR-Gas?1? signaling may prevent and/or reverse anergy. In the absence of a second stimulus, T cells activated through the TCR become unresponsive (anergic) and do not transcribe IL-2 when re-stimulated with antigen. Decreased TCR-stimulated IL-2 in T cells from SLE patients contributes to the disease process and may be due to anergy. To investigate a role for A2AR-Gas?1? signaling in anergy, the following hypotheses will be tested. (A) Gallein and/or A2AR antagonists may prevent and/or reverse anergy in T cells. (B) Gallein, ?1 siRNA, and/or A2AR antagonists may restore normal TCR-stimulated IL-2 levels in T cells from patients with SLE.

Public Health Relevance

Inflammation must be inhibited in order to treat sepsis and autoimmune diseases, whereas increased inflammation can be useful for immunotherapy of tumors and vaccine development. Current treatments have limited efficacy as well as side effects. The goal of the proposed studies is to expand on preliminary experiments that suggest that G protein ?? subunits could be useful drug targets for the diagnosis and treatment of immune system disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050369-18
Application #
8449287
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
1995-01-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2015-03-31
Support Year
18
Fiscal Year
2013
Total Cost
$326,890
Indirect Cost
$124,356
Name
Geisinger Clinic
Department
Type
DUNS #
079161360
City
Danville
State
PA
Country
United States
Zip Code
17822
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Hynes, Thomas R; Yost, Evan; Mervine, Stacy et al. (2008) Multicolor BiFC analysis of competition among G protein beta and gamma subunit interactions. Methods 45:207-13
Sheridan, Douglas L; Berlot, Catherine H; Robert, Antoine et al. (2002) A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction. BMC Neurosci 3:7