Adenosine does not bind to the growth hormone secretagogue receptor type-1a (GHS-R1a).
Ghrelin regulates GH secretion and energy homeostasis through the GH secretagogue receptor type-1a (GHS-R1a). This G-protein coupled receptor shows the peculiarity to transduce information provided not just by ghrelin as well as by adenosine through a supposed binding site different from the characterized ghrelin-binding pocket. Indeed, adenosine triggers intracellular calcium rise through a distinct signaling pathway to the one described for ghrelin, although it fails to stimulate GH secretion. Despite multiple active conformations of GHS-R1a, suggested as an explanation for a ligand-dependent activation of the downstream signaling, the concept of adenosine as agonist for GHS-R1a has been re-evaluated. The results revealed that calcium rise of both ghrelin and adenosine appears to be mediated by receptors that did not show the same sensitivity to protein kinase C (PKC) activity in GHS-R1a-transfected HEK 293 cells (HEK-GHS-R1a cells). The binding analyses showed the same number of adenosine-binding sites in both HEK 293 (B(max) = 2.01 +/- 0.15 fmol/cell) and HEK-GHS-R1a cells (B(max) = 1.90 +/- 0.11 fmol/cell). This binding was unaltered by different GHS-R1a antagonists. Western blot analysis showed a similar endogenous expression of endogenous adenosine receptor type-2b and -3 in both cell lines. The K(d) values for adenosine were 1.78 microM in HEK 293 cells and 6.30 microM in HEK-GHS-R1a cells, pointing to a modification of agonist affinity induced by overexpression of the GHS-R1a. Additionally, adenosine failed to induce the GHS-R1a endocytosis, although it attenuates the ghrelin-induced GHS-R1a endocytosis. In conclusion, adenosine is not an agonist of the GHS-R1a and its action is mediated by the endogenous adenosine receptor type-2b and -3, which is able to partially use the intracellular signaling machinery of HEK-GHS-R1a cells.
Carreira
Marcos C
MC
Laboratory of Molecular Endocrinology, Department of Medicine, University of Santiago de Compostela and Research Area, Complejo Hospitalario Universitario de Santiago (CHUS), PO Box 563, E-15780 Santiago de Compostela, Spain.
Camiña
Jesus P
JP
Díaz-Rodríguez
Esther
E
Alvear-Perez
Rodrigo
R
Llorens-Cortes
Catherine
C
Casanueva
Felipe F
FF
D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D003411:Arvicolinae; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011869:Radioligand Assay; D043704:Receptor, Adenosine A2B / Q000235:genetics; D043684:Receptor, Adenosine A3 / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D054440:Receptors, Ghrelin; D014162:Transfection / Q000379:methods
D034741:RNA, Small Interfering; D043704:Receptor, Adenosine A2B; D043684:Receptor, Adenosine A3; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D000241:Adenosine; D002118:Calcium