Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

Abstract

]substance P, a series of novel, small, peptidemimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nM agonism potency was obtained and also in one case (wFw-Isn-NH 2 , where Isn is isonipecotic acid) ϳ80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH 2 was found to be a functionally biased agonist. Thus, wFw-Isn-NH 2 mediated potent and efficacious signaling through the G␣ q and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the G␣ 12/13 pathway. The recognition pattern of wFw-Isn-NH 2 with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH 2 was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH 2 binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH 2 is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.Ghrelin is a neuroendocrine hormone that differs from other peptide hormones by a fatty acid modification, which is crucial for both the binding and activation of its receptor (1). Ghrelin is synthesized mainly in the gastrointestinal tract, where the gene coding for the peptide sequence is expressed together with the enzyme responsible for the acylation of the fatty acid to the ghrelin peptide sequence (2, 3). Multiple functions have been described for ghrelin since it was discovered. Initially it was believed that growth hormone secretion induced by ghrelin receptors in the hypothalamus and the pituitary was the primary function of ghrelin (4). However, the function of ghrelin in the hypothalamus, and in particular in the arcuate nucleus, has become the focus of attention over the last decade. In the arcuate nucleus, ghrelin is responsible for increased activity in the NPY (neuropeptide Y) and AGRP (Agouti-related protein) neurones leading to increased appetite, decreased energy expenditure, and fat accumulation (5, 6). High receptor expression is also observed in the ventromedial nucleus of the hypothalamus, and the function in this area has been proposed to be orexigenic based on the regulation of fatty acid metabolism (7). More recently it has been demonstrated that ghrelin is also involved in reward-seeking behavior such as alcohol and ...

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling

“…VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 sions were drawn from indirect measurement of SRE (25,52) and Rho kinase activation or by the use of a dominant negative mutant of G ␣13 (51). Our data confirm more directly that ghrelin stimulates GHS-R1a-mediated G 13 activation.…”

“…We took advantage here of G protein activation BRET biosensors (31,32) that directly report on the conformational change of G protein upon activation, to assess whether the partial agonist behavior of these ligands toward IP1 production resulted from their G q partial agonism. Although it was reported that GHSR1a activated G protein-dependent signaling pathways through G q , G i , G o (23,30,50), and G 13 (51), these conclusions were drawn from studies that indirectly measured G protein activation. We monitored here the selective coupling of GHS-R1a to the G protein family using activation biosensors for a panel of G protein subtypes and isoforms (G q , G i1 , G i2 , G i3 , G oa , G ob , G s , G 12 , and G 13 ).…”

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

“…Although a few ligands have already been described for the ghrelin receptor, exhibiting some signaling bias, none has been associated with ␤-arrestin. The ghrelin peptide mimetic agonist wfw-Isn-NH 2 supports receptor internalization, G q/11 signaling, and ERK1/2 phosphorylation but not SRE-mediated transcriptional activity or food intake (27). GSK161443, a small molecule ghrelin antagonist of calcium mobilization and inositol phosphate turnover (58), acts in vivo as an antagonist of GH release, an inducer of food intake, and a promoter of increased body weight (59,60), but the mechanistic and physiological basis for this has not been determined.…”

“…G q/11 and G i/o both mediate Ca 2ϩ release, and G q/11 signaling leads to ERK activation (45), but it is unclear whether G i/o coupling to GHSR1a has the same effect. G q/11 and G 12/13 , but not G i/o , activate SRE transcription downstream of GHSR1a (27), but the contribution of ␤-arrestin is, again, unknown. To measure this signaling, we employed two bioluminescence-based luciferase reporter assays, an SRE assay for MAPK/ERK1/2 activation and an SRF-RE assay, a modified SRE for RhoA/ROCK activation (46,47).…”

“…G i/o signaling has been also reported for GHSR1a in GTP␥S assays of cell membranes overexpressing GHSR1a (24) and in lipid disc assays (25). The signaling cascades downstream of GHSR1a are not well studied, but GHSR1a exerts a proliferative effect through activation of the MAPK/ERK and IRS-1 signaling cascades in several cellular systems (26) and activates the small GTPase RhoA (27).…”