Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor.
Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat (r) GHRH-R-transfected BHK cells, with the GHRH agonist, [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH(2) (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GHRH-R was blocked by phenyl arsine oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-transfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-dependent internalization. Experiments in anterior pituitary using Bodipy-FL-C(5) ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Co-localization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-R was maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR).
Veyrat-Durebex
Christelle
C
Laboratory of Neuroendocrinology of Aging, Centre Hospitalier de l'Université de Montréal Research Center, Notre-Dame Hospital and Department of Medicine, University of Montreal, Montreal, Quebec, Canada H2L 4M1.
Pomerleau
Luc
L
Langlois
Daniel
D
Gaudreau
Pierrette
P
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000378:metabolism*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001152:Arsenicals; C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; C029341:oxophenylarsine; D013007:Growth Hormone-Releasing Hormone; C048250:somatotropin releasing hormone receptor