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. 2022 Sep;179(18):4473-4485.
doi: 10.1111/bph.15866. Epub 2022 Jun 8.

N-terminal alterations turn the gut hormone GLP-2 into an antagonist with gradual loss of GLP-2 receptor selectivity towards more GLP-1 receptor interaction

Maria Buur Nordskov Gabe  1 , Laerke Smidt Gasbjerg  1 , Sarina Gadgaard  1 , Peter Lindquist  1 , Jens Juul Holst  1   2 , Mette Marie Rosenkilde  1
Affiliations

N-terminal alterations turn the gut hormone GLP-2 into an antagonist with gradual loss of GLP-2 receptor selectivity towards more GLP-1 receptor interaction

Maria Buur Nordskov Gabe et al. Br J Pharmacol. 2022 Sep.

Abstract

Background and purpose: To fully elucidate the regulatory role of the GLP-2 system in the gut and the bones, potent and selective GLP-2 receptor (GLP-2R) antagonists are needed. Searching for antagonist activity, we performed systematic N-terminal truncations of human GLP-2(1-33).

Experimental approach: COS-7 cells were transfected with the human GLP-2R and assessed for cAMP accumulation or competition binding using 125 I-GLP-2(1-33)[M10Y]. To examine selectivity, COS-7 cells expressing human GLP-1 or GIP receptors were assessed for cAMP accumulation.

Key results: Affinity of the N-terminally truncated GLP-2 peptides for the GLP-2 receptor decreased with reduced N-terminal peptide length (Ki 6.5-871 nM), while increasing antagonism appeared with inhibitory potencies (IC50 ) values from 79 to 204 nM for truncation up to GLP-2(4-33) and then declined. In contrast, truncation-dependent increases in intrinsic activity were observed from an Emax of only 20% for GLP-(2-33) up to 46% for GLP-2(6-33) at 1 μM, followed by a decline. GLP-2(9-33) had the highest intrinsic efficacy (Emax 65%) and no antagonistic properties. Moreover, with truncations up to GLP-2(8-33), a gradual loss in selectivity for the GLP-2 receptor appeared with increasing GLP-1 receptor (GLP-1R) inhibition (up to 73% at 1 μM). Lipidation of the peptides improved antagonism (IC50 down to 7.9 nM) for both the GLP-2 and the GLP-1R.

Conclusion and implications: The N-terminus of GLP-2 is crucial for GLP-2R activity and selectivity. Our observations form the basis for the development of tool compounds for further characterization of the GLP-2 system.

Keywords: GLP-1 receptor; GLP-2; GLP-2 receptor; GPCR; N-terminus; antagonists; family B1 GPCR.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. MBNG, LSG, JJH and MMR are co‐founders of Antag Therapeutics ApS. MMR and JJH are also co‐founders of Bainan Biotech ApS.

Figures

FIGURE 1
FIGURE 1
N‐terminally truncated GLP‐2 variants on the human GLP‐2R. (a) Schematic overview of the N‐terminally truncated GLP‐2 variants. The black spiral indicates the predicted α‐helix structure from amino acid number 4 to amino acid number 29 (34). COS‐7 cells were transiently transfected with the human GLP‐2R and the N‐terminally truncated GLP‐2 variants competed with [125I]‐GLP‐2(1‐33)[M10Y] giving the (b) log IC50 values calculated from (c) the inhibition curves. The dashed line in (c) represents human GLP‐2. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate. (d) A representation of the human GLP‐2R structure (grey—PDBid: 7D68) in complex with the full length GLP‐2 peptide (green) is showed where the interactions between the N‐terminal GLP‐2 residues and the GLP‐2R are highlighted.
FIGURE 2
FIGURE 2
cAMP activation and inhibition profiles of N‐terminally truncated GLP‐2 variants on the human GLP‐2R. COS‐7 cells were transiently transfected with human GLP‐2R and assessed for either cAMP activation or inhibition upon stimulation with the GLP‐2 truncated variants of (a) GLP‐2(2‐33), (b) GLP‐2(3‐33), (c) GLP‐2(4‐33), (d) GLP‐2(5‐33), (e) GLP‐2(6‐33), (f) GLP‐2(7‐33), (g) GLP‐2(8‐33), (h) GLP‐2(9‐33), (i) GLP‐2(10‐33) or (j) GLP‐2(11‐33). The dashed line in each graph represents human GLP‐2. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate
FIGURE 3
FIGURE 3
cAMP activation and inhibition profiles of GLP‐2(3‐33)[D3A] on the human GLP‐2R. (a) Schematic overview of GLP‐2(3‐33)[D3A]. The black dot indicates where the amino acid has been substituted. COS‐7 cells were transiently transfected with the human GLP‐2R and assessed for (b) competition with [125I]‐GLP‐2(1‐33)[M10Y] or (c) cAMP activation or inhibition with GLP‐2(3‐33)[D3A]. The dashed line represents human GLP‐2. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate
FIGURE 4
FIGURE 4
Schild plots of GLP‐2(2‐33), GLP‐2(3‐33), GLP‐2(3‐33)[D3A] and GLP‐2(4‐33) on the human GLP‐2R. COS‐7 cells were transiently transfected with the human GLP‐2R and assessed for cAMP accumulation upon ligand stimulation with GLP‐2 in the absence or presence of increasing concentrations (a) GLP‐2(2‐33), (b) GLP‐2(3‐33), (c) GLP‐2(3‐33)[D3A] and (d) GLP‐2(4‐33). The corresponding Schild plots of (e) GLP‐2(2‐33), (f) GLP‐2(3‐33), (g) GLP‐2(3‐33)[D3A] and (h) GLP‐2(4‐33) indicating their respective pA2 value. Data are shown as mean ± SEM, from n = 5 independent experiments carried out in duplicate.
FIGURE 5
FIGURE 5
Selectivity test of the N‐terminally truncated GLP‐2 variants on human GLP‐1R and human GIPR. COS‐7 cells were transiently transfected with either human GLP‐2R, human GLP‐1R or human GIPR, and the agonistic and antagonistic profiles of the N‐terminally truncated GLP‐2 variants were examined in cAMP accumulation. (a) Agonistic and (b) antagonistic activities of the N‐terminally truncated GLP‐2 variants on human GLP‐2R, (c) agonistic and (d) antagonistic activities of the N‐terminally truncated GLP‐2 variants on the human GLP‐1R and (e) agonistic and (f) antagonistic activites of the N‐terminally truncated GLP‐2 variants on the human GIPR. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate
FIGURE 6
FIGURE 6
cAMP activation and inhibition profiles of N‐terminally truncated and lipidated GLP‐2 variants on the human GLP‐2R. (a) Schematic overview of the N‐terminally truncated lipidated GLP‐2 variants. The red circles indicate where a 16‐carbon fatty diacid chain has been attached. COS‐7 cells were transiently transfected with human GLP‐2R and assessed for either cAMP activation or inhibition with (b) GLP‐2(3‐33)(C16‐diacid/3), (c) GLP‐2(4‐33)(C16‐diacid/4) or (d) GLP‐2(5‐33)(C16‐diacid/5). (e) Schematic overview of the N‐terminally truncated amino acid modified lipidated GLP‐2 variants. The red circles indicate where a 16‐carbon fatty diacid chain has been attached, and the black dots indicate where an amino acid has been substituted. COS‐7 cells were transiently transfected with the human GLP‐2R and assessed for either cAMP activation or inhibition with (f) GLP‐2(3‐33)[N16K](C16‐diacid/16), (g) GLP‐2(3‐33)[R20K](C16‐diacid/20) or (h) GLP‐2(3‐33)(C16‐diacid/30). The dashed line represents human GLP‐2. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate.
FIGURE 7
FIGURE 7
Selectivity test of the N‐terminally truncated and lipidated GLP‐2 variants on the human GLP‐1R. COS‐7 cells were transiently transfected with the human GLP‐2R or human GLP‐1R and assessed for either cAMP activation (a) or inhibition (b) with GLP‐2(3‐33)(C16‐diacid/3), GLP‐2(4‐33)(C16‐diacid/4) and GLP‐2(5‐33)(C16‐diacid/5) or cAMP activation (c) or inhibition (d) with GLP‐2(3‐33)[N16K](C16‐diacid/16) and GLP‐2(3‐33)[R20K](C16‐diacid/20). The full lines represent the human GLP‐1R, and the dashed lines the human GLP‐2R. Data are shown as mean ± SEM, from n = 3 independent experiments carried out in duplicate
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