FIGURE LEGENDS
Figure 1. Sequence alignment of GLP-2 and related peptides and activity of hGLP-2 and variants at the hGLP-2R. (a) Alignment of the class B1 GPCR peptides; hGLP-2(1-33), hGIP(1-42), hGCG(1-29), hGLP-1(7-36) (top panel) and the GLP-2 variants; hGLP-2(3-33), hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y) (bottom panel). In the top panel, dark grey refers to positions, which are fully conserved (identical), medium grey refers to positions with strongly similar residues, while light grey refers to positions with weakly similar residues. The red box marks position 10 (counted from residue 1 of hGLP-2(1-33)). (b-d) cAMP accumulation dose-response curve for hGLP-2R stimulated with increasing concentration of (b) hGLP-2(1-33) (n = 10) and hGLP-2(1-33,M10Y) (n =4) , (c) hGLP-2(3-33) (n = 3) and hGLP-2(3-33,M10Y) (n = 7) , and (d) hGLP-2(1-33) in the presence of 100 mM and 1 µM hGLP-2(3-33,M10Y) (n = 3) . To compensate for inter-assay variations, data have been normalized to hGLP-2(1-33) within each experiment. The experiments were carried out in duplicates and presented as mean ± SEM.
Figure 2. Homologous competition binding and binding kinetic experiments. (a, b) Homologous binding curve using (a) [125I]-hGLP-2(1-33,M10Y) (black) (n = 5)and (b) [125I]-hGLP-2(3-33,M10Y) (red) (n = 5) . To compensate for inter-assay variations, the data have been normalized to the specific binding to hGLP-2R within each assay. (c) Bmax for [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red), normalized to Bmax of [125I]-hGLP-2(1-33,M10Y). (d) Association (n = 4) and (e) dissociation (n = 4) of [125I]-hGLP-2(1–33,M10Y) (black) and [125I]-hGLP-2(3–33,M10Y) (red) on/from hGLP-2R. The dissociation was initiated by the addition of 1 μM unlabeled hGLP-2(1–33,M10Y) or hGLP-2(3–33,M10Y). (f) Comparison of binding kinetic parameters between [125I]-hGLP-2(1–33,M10Y) (black) and [125I]-hGLP-2(3–33,M10Y) (red) obtained from association and dissociation assays. To compensate for inter-assay variations data have been normalized for each radioligand within each assay. Differences were analyzed by paired t-test and significance indicated by asterisks, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and *p < 0.05. ns indicates non-significant differences. The experiments were carried out in duplicated and presented as mean ± SEM.
Figure 3. Heterologous competition binding using radiolabeled hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y). (a) Bar chart of the pIC50 values for binding of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red). (b-e) Competition binding of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red) displaced by increasing concentrations of (b) hGLP-2(1-33) (n = 5) , (c) hGLP-2(1-33,M10Y) (n = 5 for [125I]-hGLP-2(1-33,M10Y) and n = 6 for [125I]-hGLP-2(3-33,M10Y)) , (d) hGLP-2(3-33)(n = 5 for [125I]-hGLP-2(1-33,M10Y) and n = 6 for [125I]-hGLP-2(3-33,M10Y)) , and (e) hGLP-2(3-33,M10Y) (n = 5) . To compensate for inter-assay variations the data were normalized to the specific binding of hGLP-2R for each radioligand within each assay. Differences were analyzed by paired t-test and significance indicated by asterisks, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and *p < 0.05. ns indicates non-significant differences. The experiments were carried out in duplicated and presented as mean ± SEM.
Figure 4. Test for selectivity among class B1 GPCRs. (a) Phylogenetic tree of the class B1 subfamily GPCRs consisting of the GLP-2R and 14 sequence related GPCRs (modified from (Gasbjerg et al.. 2018)). (b) Heterologous binding of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red) to the hGLP-1R(n = 3) , hGIPR (n = 2) , hGCGR (n = 2) , hSecretinR(n = 2) , VPAC-1R (n = 2) and VPAC-2R (n = 2)displaced by increased concentrations of endogenous hGLP-2(1-33). The experiments were carried out in duplicated and presented as mean ± SEM.