Gonadotropin releasing hormone (GnRH) is the central neuroendocrine mediator controlling the biosynthesis and secretion of gonadotropic hormones. The GnRH receptor (R) serves as the central integrator of hypothalamic signals to coordinate gonadotrope behavior in vivo. In the absence of this critical neuroendocrine interface, reproduction ceases. The GnRHR is a unique G-protein-coupled receptor that displays constitutive plasma membrane localization in raft compartments; this membrane compartmentalization is required for efficient cell signaling to pathways critical for fertility in female mammals. In this revised application, we focus our attention on the novel identification of co-localization of the F0F1 ATP synthase complex and enzymes in the electron transport chain with the GnRHR within membrane rafts in pituitary gonadotropes. Unique preliminary proteomic studies in this exploratory R21 application have identified a suite of 129 peptides that specifically localize with the GnRHR at the plasma membrane providing a vigorous opportunity to explore how these membrane-associated complexes control GnRH action and potentially fertility. Preliminary studies in support of the proposed research plan focus on the functional importance of the F0F1 ATP synthase complex identified at the cell surface through interactions with the GnRHR and the membrane raft marker flotillin 1 in a gonadotrope cell model system. These studies demonstrate that the gonadotrope can actively synthesize and metabolize ATP extracellularly. Further, extracellular ATP tone modulates GnRH-induced secretion of LH from mouse pituitaries in explant culture. Based upon these preliminary studies, our central working hypothesis that activation of the GnRHR within membrane rafts is modulated by extracellular ATP through the actions of F0F1 ATP synthase and ectoNTPDase CD39 as paracrine effectors at the cell surface. ATP paracrine modulation of GnRH action will impact the secretion of LH. The following Specific Aims examine this central working hypothesis: Aim 1. Analyze the role of the electron transport chain in regulating cell surface F0F1 ATP synthase activity in gonadotropes. Aim 2. Determine the physiological importance of the ectoNTPDase CD39 in modulating GnRH action. Aim 3. Define the origin of F0F1 ATP synthase metabolites in the extracellular space.