Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors

• Published in: American Journal of Physiology: Cell Physiology 2009-09, Vol.297 (3), p.C591-C600
• Main Authors: Finch, Ann R, Caunt, Christopher J, Armstrong, Stephen P, McArdle, Craig A
• Format: Article
• Language: English
• Subjects: Animals; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Buserelin - pharmacology; Cancer; Cells; Down-Regulation; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; Gonadotropin-Releasing Hormone - metabolism; HeLa Cells; Hemagglutinins; Hormones; Humans; Indoles - pharmacology; Mice; Pyridines - pharmacology; Receptors and Signal Transduction; Receptors, LHRH - genetics; Receptors, LHRH - metabolism; Recombinant Proteins; Rodents; Signal Transduction; Xenopus
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00166.2009

Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, United Kingdom Submitted 17 April 2009 ; accepted in final form 4 July 2009 Gonadotropin-releasing hormone (GnRH) acts via seven transmembrane receptors to stimulate gonadotropin secretion. Sustained stimulation desensitizes GnRH receptor (GnRHR)-mediated gonadotropin secretion, and this underlies agonist use in hormone-dependent cancers. Since type I mammalian GnRHR do not desensitize, agonist-induced internalization and downregulation may underlie desensitization of GnRH-stimulated gonadotropin secretion; however, research focus has recently shifted to anterograde trafficking, with the finding that human (h)GnRHR are mostly intracellular. Moreover, there is little direct evidence for agonist-induced trafficking of hGnRHR, and whether or not type I mammalian GnRHR show agonist-induced internalization is controversial. Here we use automated imaging to monitor expression and internalization of hemagglutinin (HA)-tagged hGnRHRs, mouse (m) GnRHR, Xenopus (X) GnRHRs, and chimeric receptors (hGnRHR with added XGnRHR COOH tails, h.XGnRHR) expressed by adenoviral transduction in HeLa cells. We find that agonists stimulate downregulation and/or internalization of mGnRHR and XGnRHR, that GnRH stimulates trafficking of hGnRHR and can stimulate internalization or downregulation of hGnRHR when steps are taken to increase cell surface expression (addition of the XGnRHR COOH tail or pretreatment with pharmacological chaperone). Agonist effects on internalization (of h.XGnRHR) and downregulation (of hGnRHR and h.XGnRHR) were not mimicked by a peptide antagonist and were prevented by a mutation that prevents GnRHR signaling, demonstrating dependence on receptor signaling as well as agonist occupancy. Thus agonist-induced internalization and downregulation of type I mammalian GnRHR occurs in HeLa cells, and we suggest that the high throughput imaging systems described here will facilitate study of the molecular mechanisms involved. seven transmembrane receptor; trafficking; G protein-coupled receptor Address for reprint requests and other correspondence: C. A. McArdle, Univ. of Bristol, Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Bldg., Whitson St., Bristol, BS1 3NY, UK (e-mail: craig.mcardle{at}bris.ac.uk ).