Desensitization and endocytosis mechanisms of ghrelin-activated growth hormone secretagogue receptor 1a.
In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [(125)I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H(+)-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.
Molecular and Cellular Endocrinology Laboratory, Department of Medicine, Complejo Hospitalario Universitario de Santiago-Universidad de Santiago de Compostela, P.O. Box 56, E-15780 Santiago de Compostela, Spain.
Jesus P
JP
Camiña
Marcos C
MC
Carreira
Said
S
El Messari
Catherine
C
Llorens-Cortes
Roy G
RG
Smith
Felipe F
FF
Casanueva
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D022163:Clathrin-Coated Vesicles / Q000502:physiology; D006224:Cricetinae; D004622:Embryo, Mammalian; D004705:Endocytosis; D004789:Enzyme Activation; D054439:Ghrelin; D049452:Green Fluorescent Proteins; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D007668:Kidney; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D036361:Peptide Hormones / Q000378:metabolism / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D054440:Receptors, Ghrelin; D011993:Recombinant Fusion Proteins; D014162:Transfection
D054439:Ghrelin; D007457:Iodine Radioisotopes; D008164:Luminescent Proteins; D036361:Peptide Hormones; D019269:Phosphatidylinositol 4,5-Diphosphate; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium