Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Abstract

Liver‐expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein‐coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin–GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non‐competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half‐maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non‐competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N‐terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.

Growth hormone secretagogue receptor signalling affects high‐fat intake independently of plasma levels of ghrelin andLEAP2, in a 4‐day binge eating model

“…In summary, in the present study, we found that central administration of the GHSR antagonist [D-Lys3]-GHRP-6 or JMV2959 did not affect HF intake escalation over the successive days or cumulative 4-day HF intake, supporting the notion that circulating ghrelin does not affect brain areas regulating binge-like HF intake. By contrast, i.c.v.-injected K-(D-1-Nal)-FwLL-NH 2 reduced daily HF intake, HF intake escalation over the successive days and cumulative 4-day HF intake, in a similar fashion to that seen for i.c.v.-injected LEAP2 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . Notably, central administration of these GHSR synthetic ligands did not affect 22hour food intake and body weight.…”

“…43 Because we recently demonstrated that the N-terminal sequence of LEAP2 confers full receptor binding and activity, 7 a peptide containing the initial 14 residues of N-terminal LEAP2 sequence (hereafter named LEAP2 1-14 ) was employed as an analogue of full-length LEAP2. LEAP2 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] was assembled on solid support using Fmoc chemistry and starting from Amphisphere 40 RAM resin (Agilent Technologies Inc., Santa Clara, CA, USA), purified by reverse phase-high-performance liquid chromatography and characterised by liquid chromatography-mass spectrometry and matrix-assisted laser desorption/ionization-tandem mass spectrometry with a purity > 95%, as described previously. 7 LEAP2 1-14 was dissolved in PBS.…”

“…To test whether central administrations of ghrelin or LEAP2 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] affect HF intake in mice exposed to the binge-like eating protocol, ad lib. fed mice were daily i.c.v.-injected with aCSF alone or containing each of these peptides and subsequently exposed to a HF pellet for 4 consecutive days ( Figure 3A and (iii) a reduced cumulative 4-day HF intake.…”

“…In vitro, GHSR shows an unusually high constitutive activity that signals with approximatey 50% of its maximum activity, in the absence of ghrelin . Constitutive GHSR activity is reduced by LEAP2, indicating that this ligand also acts as an inverse agonist of the receptor . A role for constitutive GHSR activity in humans is supported by a naturally occurring mutation of GHSR that abolishes constitutive activity without altering ghrelin‐evoked activity and leads to familial short stature .…”

Liver Expressed Antimicrobial Peptide 2 is Associated with Steatosis in Mice and Humans

“…LEAP2 is primarily expressed in liver, kidney, intestine and stomach [4][5][6]. Recent evidence reveal that LEAP2 is a negative feedback modulator that acts on growth hormone secretagogue receptor (GHSR) as an endogenous ligand with inverse agonist/antagonist properties [7][8][9][10]. In 2018, Ge et al [7] firstly reported that LEAP2 functions as an endogenous antagonist of GHSR.…”