Visualization of unoccupied and occupied gonadotropin-releasing hormone receptors in living cells

• Published in: Molecular and cellular endocrinology Vol. 146; no. 1; pp. 27 - 37
• Main Authors: Lin, Xinwei, Cornea, Anda, Janovick, Jo Ann, Conn, P.Michael
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 25.11.1998
• Subjects: Animals; Buserelin - pharmacology; Catfishes; Cell Line; Cell Membrane - chemistry; Confocal microscopy; Cyclic AMP - metabolism; GnRH receptor; Green fluorescent protein; Green Fluorescent Proteins; Humans; Inositol Phosphates - metabolism; Luminescent Proteins - genetics; Microscopy, Confocal; Pituitary Gland - chemistry; Pituitary Gland - metabolism; Prolactin - metabolism; Rats; Receptors, LHRH - analysis; Receptors, LHRH - genetics; Receptors, LHRH - metabolism; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - metabolism; Signal Transduction; Transfection; Confocal microscopy; GnRH receptor; Green fluorescent protein
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/S0303-7207(98)00204-4

Three chimeras of the rat GnRH receptor (rGnRHR) and an enhanced green fluorescent protein (GFP) were assessed to examine their suitability as probes of the receptor in transfected GH 3 cells. Direct fusion of GFP to the N or C terminus of the rGnRHR abolished the receptor ligand binding affinity and the chimeric receptors were intracellularly localized. In contrast, rGnRHR–Ctail–GFP, a fusion of the N-terminus of the GFP to the C-terminus of the rGnRHR with the intracellular C-terminal tail of the catfish GnRHR as an intermediate spacer, was functional in terms of plasma membrane localization, ligand binding ability, receptor-mediated signal transduction and pattern of homologous down-regulation. The functional chimera of GnRHR and GFP provided a useful model for observation of GnRHR distribution and agonist-stimulated trafficking in living cells.