Identification of the GnRH-(1-5) signaling pathway in immortalized GnRH neurons

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-144
Darwin Omar Larco*1, Nina Semsarzadeh2, Madelaine Clark2 and Tao-Yiao John Wu3
1Uniformed Services University, Bethesda, MD, 2USUHS, Bethesda, MD, 3The Uniformed Services Universit, Bethesda, MD
The gonadotropin-releasing hormone (GnRH) is a decapeptide critical for the regulation of reproductive function and behavior. We have previously demonstrated that the GnRH metabolite, GnRH-(1-5), is biologically active and binds the G protein-coupled receptor 173 (GPR173) to inhibit the migration of GN11 cells, an immortalized GnRH-secreting cell. In this study, we investigated the downstream signaling pathway of GnRH-(1-5) involved in GN11 cellular migration. To determine whether GnRH-(1-5) regulates migration in a G protein-dependent mechanism, cells were treated with a generic G protein antagonist, G protein antagonist-2 (GPAnt2), in the presence of GnRH-(1-5) and a wound healing assay was conducted to measure migration. Interestingly, GPAnt-2 treatment abolished the GnRH-(1-5) inhibition of migration, indicating the mechanism of GnRH-(1-5) is G protein-coupled. Next, we measured the second messengers cyclic adenosine monophosphate (cAMP) and inositol triphosphate (IP3) levels to identify the potential Gα subunit recruited by the association of GnRH-(1-5) and GPR173. GnRH-(1-5) treatment did not alter cAMP levels relative to cells treated with vehicle (VEH) or forskolin, suggesting GnRH-(1-5) does not couple to the Gαs or Gαi subunits. Similarly, IP3 levels were unchanged by GnRH-(1-5) treatment, indicating a Gαq/11-independent mechanism. In the absence of GnRH-(1-5) activating these second messenger systems, it likely the interaction of GnRH-(1-5) with GPR173 recruits G proteins important in cytoskeletal rearrangement. Much of this signaling can occur within lipid rafts of the plasma membrane. To determine whether GPR173 colocalizes with known markers of lipid rafts, immunofluorescent and subcellular fractionation studies were conducted in GN11 cells. Interestingly, we found GPR173 colocalizes with caveolin-1 and flotillin-1, indicating that this receptor associates with lipid rafts. In conclusion, we show that the GnRH-(1-5) and GPR173 mechanism is complex by deviating from the canonical GPCR signaling pathway. Identification of the GnRH-(1-5) mechanism may lie within lipid rafts, dense regions of signaling within the plasma membrane, to inhibit the cytoskeletal machinery driving the migration of GN11 cells.

Nothing to Disclose: DOL, NS, MC, TYJW

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: National Science Foundation Grant IOS-1052288 and DOD RO85FN