title: β-Arrestin 2 is a mediator of GnRH-(1-5) signaling in immortalized GnRH neurons.
abstract: We have previously demonstrated that the cleavage product of the full-length GnRH, GnRH-(1-5), is biologically active, binds G protein-coupled receptor 173 (GPR173), and inhibits the migration of cells in the immortalized GnRH-secreting GN11 cell. In this study, we attempted to characterize the GnRH-(1-5) intracellular signaling mechanism. To determine whether the signaling pathway mediating GnRH-(1-5) regulation of migration involves a G protein-dependent mechanism, cells were treated with a generic G protein antagonist in the presence and absence of GnRH-(1-5), and a wound-healing assay was conducted to measure migration. G Protein antagonist 2 treatment abolished the GnRH-(1-5) inhibition of migration, indicating that the mechanism of GnRH-(1-5) is G protein coupled. To identify the potential Gα-subunit recruited by GnRH-(1-5) binding GPR173, we measured the second messengers cAMP and inositol triphosphate levels. GnRH-(1-5) treatment did not alter cAMP levels relative to cells treated with vehicle or forskolin, suggesting that GnRH-(1-5) does not couple to the Gαs or Gαi subunits. Similarly, inositol triphosphate levels remained unchanged with GnRH-(1-5) treatment, indicating a mechanism not mediated by the Gαq/11 subunit. Therefore, we also examined whether GnRH-(1-5) activating GPR173 deviated from the canonical G protein-coupled receptor signaling pathway by coupling to β-arrestin 1/2 to regulate migration. Our coimmunoprecipitation studies indicate that GnRH-(1-5) induces the rapid interaction between GPR173 and β-arrestin 2 in GN11 cells. Furthermore, we demonstrate that this association recruits phosphatase and tensin homolog to mediate the downstream action of GnRH-(1-5). These findings suggest that the GnRH-(1-5) mechanism deviates from the canonical G protein-coupled receptor pathway to regulate cell migration in immortalized GnRH neurons.
authors.affiliation: PhD, Department of Obstetrics and Gynecology, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, Maryland 20814. tao-yiao.wu@usuhs.edu.
authors.forename: Darwin O
authors.initials: DO
authors.lastname: Larco
authors.forename: Nina N
authors.initials: NN
authors.lastname: Semsarzadeh
authors.forename: Madelaine
authors.initials: M
authors.lastname: Cho-Clark
authors.forename: Shaila K
authors.initials: SK
authors.lastname: Mani
authors.forename: T John
authors.initials: TJ
authors.lastname: Wu
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D000242:Cyclic AMP; D007987:Gonadotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605255:Arrb1 protein, mouse; C000605258:Arrb2 protein, mouse; D019390:Arrestins; C578985:GPR173 protein, mouse; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D007987:Gonadotropin-Releasing Hormone; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP