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.