SAT-007 Proteomic analysis reveals cell adhesion associated proteins as effectors ofgonadotropin releasing hormone receptor signaling

Abstract Disclosure: H. Alqahtani: None. ABSTRACT Stimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anteriorpituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. Oneimportant downstream component of GnRH receptor signaling is activation of the mitogen-activatedprotein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of thegonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasmamembrane domains where they participate in multiprotein signaling complexes. Here we usedquantitative proteomics to identify proteins associated with low-density plasma membrane domains andto measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-densityplasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination withmass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins withthis plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actincytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and thesmall GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstratingfunctional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. In sum, these dataindicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essentialeffector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our data show thatacute stimulation of GnRH receptors alters the fractional membrane abundance of proteins, such as actincytoskeletal-related proteins, mechanistically linked to gonadotrope activation. Presentation: Saturday, July 12, 2025