EGFR transactivation and enhanced cell migration by Gonadotropin-Releasing Hormone (GnRH) Metabolite, GnRH-(1-5), is Mediated by GPR101 in the Ishikawa Cell Line

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-147
Madelaine J Cho-Clark*, Darwin Omar Larco, Nina Samsarzadeh and Tao-Yiao John Wu
Uniformed Services University, Bethesda, MD
GnRH is a decapeptide known for its key role in regulating the hypothalamic-pituitary-gonadal (HPG) axis.  Although its major role has been well characterized, its secondary level of regulation in peripheral tissues remains unclear.  GnRH is processed by zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr5-Gly6 bond to form a pentapeptide, GnRH-(1-5); this pentapeptide has alternate actions from its parent peptide GnRH in both neural and peripheral cells.  Here, we wish to determine the effect of GnRH-(1-5) on cellular migration through the epidermal growth factor receptor (EGFR).  Using the Ishikawa cell line as a model of endometrial cancer, we have previously shown that GnRH-(1-5) increases the phosphorylation of EGFR (p<0.05) at three tyrosine sites (Y992, Y1045, Y1068).  This activation of EGFR by GnRH-(1-5) was associated with rapid stimulation of EGF release, suggesting GnRH-(1-5) promotes the transactivation of EGFR.  To investigate whether EGFR transactivation by GnRH-(1-5) is mediated by GnRH-(1-5) receptors, we performed a high-throughput β-Arrestin recruitment assay screening for binding to orphan G-protein coupled receptors (GPR).  Among the 4 GPRs identified, GPR101 expression was found to selectively mediate GnRH-(1-5) action.  Down-regulation of GPR101 expression by antisense oligonucleotides blocked GnRH-(1-5) mediated EGF release and phosphorylation of EGFR at all 3 tyrosine residues.  To determine whether GnRH-(1-5) and its parental analogs had an effect on migration in the cell line, the in vitro scratch wound assay was utilized.  Treatment with 100nM GnRH-(1-5) increased migration into the wound area by ~20% (p<0.05) and ~45% (p<0.05) at t= 24 and 48h respectively when compared to VEH.  Conversely, LHRH analogs D-Ser-LHRH and D-Trp-LHRH suppressed migration into the wound area.  Down-regulation of GPR101 expression by antisense oligonucleotides blocked GnRH-(1-5) migration effects.  Furthermore, treatment of cells with a G-protein antagonist, GPAnt2 and the EGFR antagonist, AG1478, blocked the GnRH-(1-5) facilitated increase in cellular migration.  In summary, our results suggest that GnRH-(1-5) stimulated migration is dependent on GPR101 in a manner dependent on G-proteins and transactivation of EGFR.

Nothing to Disclose: MJC, DOL, NS, 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: NSF IOS-1052288; DoD RO85FN