Characterization of an Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptor and Effects of Ligand Binding on Receptor Lateral Diffusion*

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotropederived aT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly,D-Ala--GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in aT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2–1.6 3 10 cm/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76–91% to 44– 61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist. (Endocrinology 140: 950–957, 1999) S the elucidation of the primary amino acid sequence of GnRH over 25 yr ago, a clear picture of the key role of this molecule in controlling the reproductive function of mammals has emerged. The pulsatile discharge of GnRH from hypothalamic neurosecretory neurons not only stimulates but is obligatory for the synthesis and secretion of LH, and to a lesser extent FSH, from the anterior pituitary gland (1, 2). Not surprisingly, changes in GnRH secretory rates are a primary regulator of gonadotropin secretion (2). Similarly, changes in the number of pituitary receptors for GnRH have been implicated as an important mechanism underlying the regulation of gonadotropin secretion (3–5). Thus, changes in pituitary content and secretion of LH may not only depend on changes in GnRH availability but also on the number of GnRH receptors (GnRHR) available for binding and consequently the responsiveness of gonadotropes to GnRH (6). With the recent availability of complementary DNAs (cDNAs) encoding the GnRHR (7–11), researchers have found that changes in the number of GnRHRs are correlated with concomitant fluxes in steady state concentrations of messenger RNA for the GnRHR (5, 12–15), thus implicating transcriptional regulation as an important component underlying the number of GnRHRs in the pituitary gland. Consistent with this, we have found that the promoter of the GnRHR gene is hormonally regulated in transgenic mice (16). Not only has the GnRHR cDNA proven useful as a probe for gene expression, but it has also allowed investigations of the structure-function relationships that exist in this protein. In this regard, the cDNA for the murine GnRHR predicts a protein containing seven hydrophobic amino acid domains and suggests membership of the GnRHR in the G proteincoupled receptor superfamily (7), a classification that is consistent with the proposed mechanism(s) of action of GnRH (15, 17). The predicted GnRHR structure is, however, somewhat unusual in that it possesses an extremely short intracellular carboxyl-terminus of only one or two amino acids. This structural difference alone raises interesting questions about the functional domains of this molecule required for trafficking, signal transduction, and internalization. In fact, significant progress has been made in dissecting the functional domains of the GnRHR (15, 17); however, large gaps remain in our understanding of this molecule. In particular, the absence of any antibodies capable of recognizing the GnRHR in situ has precluded any analyses of the GnRHR in the unbound state. Thus, it has been difficult to monitor the rate of GnRHR biosynthesis, its trafficking through various intracellular compartments, and its behavior in the plasma membrane. Nonetheless, these steps have been implicated as Received May 19, 1998. Address all correspondence and requests for reprints to: Dr. Colin M. Clay, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523. E-mail: cclay@cvmbs.colostate. edu. * This work was supported by a grant from the Colorado State University Experiment Station and NIH Grant R-29-HD-32416. † These authors contributed equally to this work. 0013-7227/99/$03.00/0 Vol. 140, No. 2 Endocrinology Printed in U.S.A. Copyright © 1999 by The Endocrine Society