Interaction between ghrelin and the ghrelin receptor (GHS-R1a), a NMR study using living cells.

The study of the interaction of ghrelin (1), the endogenous ligand for the GH secretagogues receptor (GHS-R1a), and des-acyl ghrelin (2) with the GHS-R1a by NMR using living cells is presented, using GHS-R1a stably transfected cell lines (CHO and HEK 293) and wild type cells. Therefore, the interaction of 1 and 2 with the GHS-R1a receptor has been performed using quasi-physiological conditions. Ghrelin (1), showed a higher number of residues affected by chemical shift perturbation (CSP) or chemical shift exchange (CSE) effects: Ser3, Phe4, Leu5, Val12, Gln13/Gln14, Lys16/Lys19, Glu17 and Lys24 were much more affected in 1 than in des-acyl ghrelin (2). The chemical shift index CSI values indicated the presence of a possible alpha-helical region between Glu8 and Lys20 for ghrelin (1). After analysing the NMR data, two possible structures have arisen, which present different proline rotamers: the EEZE and the EZEZ conformers, at positions Pro7, Pro21, Pro22 and Pro27, respectively, keeping a left-handed alpha-helix from Glu8 to Lys20. These experimental evidences might imply that the GHS-R1a receptor is acting as a prolyl-cis/trans isomerase.

Obestatin stimulates Akt signalling in gastric cancer cells through beta-arrestin-mediated epidermal growth factor receptor transactivation.

Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of G(i)(/o)-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.

Role of obestatin on growth hormone secretion: An in vitro approach.

Obestatin, the ghrelin-associated peptide, showed to activate MAPK signaling with no effect on Akt nor cell proliferating activity in rat tumor somatotroph cells (growth cells, GC). A sequential analysis of the obestatin transmembrane signaling pathway indicated a route involving the consecutive activation of G(i), PI3k, novel PKCepsilon, and Src for ERK1/2 activation. Furthermore, obestatin treatment triggers growth hormone (GH) release in the first 30min, being more acute at 15min. At 1h, obestatin treated cells showed the same levels in GH secretion than controls. Added to this functionality, obestatin was secreted by GC cells. Based on the capacity to stimulate GH release from somatotroph cells, obestatin may act directly in the pituitary through an autocrine/paracrine mechanism.

Lysophosphatidic acid inhibits ghrelin secretion in the human gastric adenocarcinoma AGS cell line: role of mitogenic activated protein kinase signaling pathway.

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), is a 28 amino acid residue with a post-translational octanoyl modification on Ser3. Despite the biomedical interest in this hormone, the fine details of its regulation and the mechanisms controlling its secretion are largely unknown. The present study analyzes the molecular steps involved in the full lysophosphatidic acid (LPA) receptor-mediated activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and its consequent role as an inhibitor of ghrelin secretion in the gastric adenocarcinoma cell line AGS. ERK1/2 phosphorylation mediated by LPA proceeds via activation of the type 2 LPA receptor, activation of the nonreceptor tyrosine kinase c-Src, and subsequent transactivation of the epidermal growth factor receptor. Furthermore, LPA-induced ERK activation was found to be independent of matrix metalloproteinases; thus, c-Src acted as the scaffold-transactivating epidermal growth factor receptor. Finally, a correlation was observed between the mitogenic effects of LPA and ghrelin secretion in the human gastric adenocarcinoma cell line AGS. These data suggest a possible physiological role of LPA in ghrelin secretion. The relationship found between LPA and ghrelin secretion might explain the low circulating levels of ghrelin observed in obese patients, as a bona fide reflex of the energetic stores.

Stimulation of extracellular signal-regulated kinases and proliferation in the human gastric cancer cells KATO-III by obestatin.

Obestatin, the ghrelin-associated peptide, activates cell proliferation in the gastric cancer cell line KATO-III. The results showed that this peptide induced cell proliferation by mitogen-activated kinase kinase/extracellular signal-regulated kinases1/2 (ERK1/2) phosphorylation. A sequential analysis of the obestatin transmembrane signalling pathway indicated that the ERK1/2 activity is partially blocked after preincubation of the cells with pertussis toxin, as well as by wortmannin (an inhibitor of phosphoinositide 3-kinase (PI3K)), staurosporine (an inhibitor of protein kinase C (PKC)) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon- and theta-isoenzymes rise with similar time-courses, from which PKCepsilon appears to be the responsible for ERK1/2 response. Based on the experimental data, a signalling pathway involving the consecutive activation of G(i), PI3K, novel PKCepsilon and Src for ERK1/2 activation is proposed. These results point to a functionally active peptide that regulates proliferation of the gastric cancer cells KATO-III.