Growth Hormone Secretagogues and Growth Hormone Releasing Peptides Act As Orthosteric Super-Agonists but Not Allosteric Regulators for Activation of the G Protein Gαo1 by the Ghrelin Receptor

Abstract

Some growth hormone secretagogues act as agonists at the ghrelin receptor and have been described as "ago-allosteric" ligands because of an ability to also modulate the maximum efficacy and potency of ghrelin (Holst et al., 2005). In membranes prepared from cells coexpressing the human ghrelin receptor and, growth hormone-releasing peptide 6 (GHRP-6), and the 2(R)-hydroxypropyl derivative of 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo- -692,585) each functioned as direct agonists, and each displayed higher efficacy than ghrelin. The effect of multiple, fixed concentrations of each of these ligands on the function and concentration-dependence of ghrelin and the effect of multiple, fixed concentrations of ghrelin on the action of MK-677, GHRP-6, and L-692,585 was analyzed globally according to a modified version of an operational model of allosterism that accounts for allosteric modulation of affinity, efficacy, and allosteric agonism. Each of the data sets was best fit by a model of simple competition between a partial and a full agonist. Both positive and negative allosteric modulators are anticipated to alter the kinetics of binding of an orthosteric agonist. However, none of the proposed ago-allosteric regulators tested had any effect on the dissociation kinetics of 125 I-[His]-ghrelin, and GHRP-6 and MK-677 were able to fully displace 125 I-[His]-ghrelin from the receptor. At least in the system tested, each of the ligands acted in a simple competitive fashion with ghrelin as demonstrated by analysis according to a model whereby ghrelin is a partial agonist with respect to each of the synthetic agonists tested.

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

“…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 ).…”

“…D, G 12 activation by the thromboxane A 2 ␣ type (TP␣) receptor: HEK293T cells co-expressing the TP␣ receptor and G 12 biosensor were stimulated by 10 Ϫ6 M U46619, and the BRET signal was recorded at described in A. 50 and E max are reported in Table 2. tin2 recruitment, and ERK1/2 phosphorylation. 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.…”

“…Ghrelin, MK-0677, and JMV 1843 displayed similar potency and efficacy for both G q activation and IP1 with MK-0677 displaying a higher efficacy and being more potent than JMV 1843 and ghrelin at both pathways (Fig. 5 and Table 2 for EC 50 and E max values). Quantification of bias demonstrated no selectivity of MK-0677 and JMV 1843 toward G q activation and IP1 production compared with the reference ligand ghrelin (Table 3).…”

“…In these assays, ligand concentrations of 1 M and 1:10 receptor/G protein molar ratios with receptor concentrations in the 20 nM range were used. 50 and E max values were estimated from dose-response curves using the nonlinear curve fitting equation (three parameters) in GraphPad Prism (Version 5.0).…”

“…Quantification of bias could be done for JMV 1843 and MK-0677 only and indicated that these ligands were not significantly biased toward G q activation and IP1 production relative to ␤-arrestin2 recruitment and ERK1/2 phosphorylation (Table 3). 50 and E max values were determined from dose-response curves performed in HEK293T cells expressing GHS-R1a as described under "Experimental Procedures." For the agonist and neutral compounds, E max is expressed as the percentage of maximal ghrelin.…”