The interaction of the hypothalamic peptide termed gonadotropin releasing hormone (GnRH) with specific receptors located on gonadotrope cells of the anterior pituitary gland represents a central point for regulation of reproductive function. Accordingly, much effort has been devoted toward understanding the physiological consequences of regulation of GnRH and the GnRH receptor.
One of our primary research goals is to define the molecular mechanisms underlying transcriptional regulation of the GnRH receptor gene. Toward this end, we have cloned the gene encoding the GnRH receptor and have found that cell-specific expression of this gene is mediated by a complex enhancer whose components include a binding site for the nuclear orphan receptor steroidogenic factor-1 (SF-1), a canonical AP-1 element, and an element we have termed the GnRH receptor activating sequence or GRAS. In addition to mediating cell-specific expression, this complex enhancer also integrates multiple endocrine inputs.
Research is currently underway to establish the signal transduction cascades and downstream targets that ultimately lead to GnRH activation at AP-1. Recent studies suggest that GnRH communicates with the GnRH receptor gene via two mitogen activated protein (MAP) kinase pathways (Jun N-terminal kinase or JNK and extracellular signal-regulated kinase or ERK). In a similar vein, we have recently discovered that GRAS functionally co-localizes with activin regulation of GnRH receptor gene expression. Current studies are underway to identify the protein(s) that mediate functional activity at GRAS. In fact, our most recent data suggest that GRAS may represent a composite regulatory element whose functional activity is dependent on a distal binding site for members of the SMAD family of TGF/activin signaling proteins and a proximal binding site for an as yet unidentified non-SMAD partner.
Much of our work depends on analyses of promoter function in permanent cell lines; however, we recognize the limitations of these experiments. Thus, whenever possible, we are committed to expanding our studies to a more physiological setting such as transgenic mice. In collaboration with Dr. Richard Bowen, we have established lines of transgenic mice harboring GnRH receptor fusion genes. In our view, these animals provide the most robust physiological test of transcriptional mechanisms defined in vitro. For example, with the assistance of Dr. Terry Nett, we have established specific domains of the GnRH receptor gene promoter that are necessary for both GnRH, estrogen and inhibin responsiveness in vivo.
Recent publication: White BR, Duval DL, Mulvaney JM, Robertson MS, Clay CM. 1999. Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element. Mol Endocrinol 13:566-577.
Our goals are to understand the GnRH receptor at both the genetic and protein level. Toward the latter, we have constructed functional GnRH receptors in which green fluorescent protein (GFP) is fused to the intracellular carboxyl terminus. This fusion receptor is appropriately trafficked to the plasma membrane of multiple cell-types, binds GnRH with an affinity similar to the wild-type receptor, and is capable of signal transduction. These intrinsically fluorescent molecules have afforded us a unique opportunity to study the GnRH receptor as both an occupied and unoccupied receptor. In collaboration with Dr. Deborah Roess, we are applying laser-optical techniques to study how potent GnRH agonists and antagonists alter both biophysical and biochemical attributes of the GnRH receptor in living cells.
As shown in the image above, the GnRH receptor-GFP fusion protein is targeted to the plasma membrane. Panels A, B and C show the same cell under different fluorescent illuminations. Red fluorescence is from rhodamine-concanavalin A (used to mark the plasma membrane; panel A) and green fluorescence is due to the presence of the GFP fusion protein (panel B). Panel C is a composite in which pixels that have both red and green signals are colored yellow, indicating colocalization of the plasma membrane marker and GnRHR-GFP fusion protein.
Recent publication: Nelson S, Horvat RD, Malvey J, Roess DA, Barisas BG, Clay CM. 1998. Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology 140:950-957.