Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor

✍ Scribed by Christelle Veyrat-Durebex; Luc Pomerleau; Daniel Langlois; Pierrette Gaudreau

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 347 KB
• Volume: 203
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.20233

✦ Synopsis

Abstract

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^α^‐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). © 2004 Wiley‐Liss, Inc.