DISCUSSION
Despite the emerging evidence for the biological importance of GLP-2 as a trophic hormone for the gut and bones, very little structural information is available of the GLP-2R. An increasing number of high-resolution structures of class B1 GPCRs, have been published (Liang et al. 2018; Qiao et al. 2020; Wu et al. 2020; Zhang et al. 2018; Zhang et al. 2017; Zhao et al. 2020), yet the structure of the GLP-2R remains to be determined. However, a handful of studies focusing on the GLP-2 structure and its interaction with the GLP-2R have elucidated structural requirements for GLP-2’s interaction with its receptor. In 2000, DaCambra et. al. performed an Alanine(Ala)-scan within the DPP-4 resistant h[Gly2]GLP-2(1-33) and showed reduced receptor activation (cAMP accumulation), of the rGLP-2R by alterations in the N-terminus part of the peptide (Dacambra et al. 2000). Here, Ala replacement of the Histidine1 and the Asparticacid3 of hGLP-2, severely reduced receptor activation with only modest changes in binding affinity. These data demonstrate the importance of the GLP-2 N-terminus for receptor activation, as also illustrated by the partial agonism (and competitive antagonism) of GLP-2(3-33) (Thulesen et al. 2002) (figure 1). Ala-scan within the C-terminus part of h[Gly2]GLP-2 severely reduced the binding affinity demonstrating a central role of the C-terminus part for receptor binding. In 2011, Venneti et. al. presented the first three-dimensional solution structure of GLP-2 by nuclear magnetic resonance (NMR) (Venneti et al. 2011). This structure supported the distinct roles of the N- and C-terminus part of GLP-2 and revealed a stable alpha-helical conformation at the central region (between Phe6 and Ile27) and a less well-defined helical conformation in the C-terminus region. The binding interface with the extracellular domain (ECD) of the receptor was predicted to be between Leucine17 and Lysine30, while the N-terminus part of GLP-2 from Histiine1 to Aspargine16 lacked contact with the extracellular domains of the GLP-2R. The central roles of the N- and C-terminus part of GLP-2 in respectively, receptor activation and receptor binding were supported by Yamazaki et. al. in 2013 (Yamazaki et al. 2013), showing a decreased intrinsic placental alkaline phosphatase (PALP) activity (driven by cAMP) for GLP-2(3-33), (6-33) and (11- to 13-33). Most recently, Wisniewski et. al. replaced each residue in the DPP-4 resistant [Gly2,Nle10]hGLP-2(1-30) analog with its d-enantiomer in a systematic approach to gain insight into the GLP-2R recognition revealing a loss of potency at position 5, 8, 9, 12, and 14 in the N-terminus, and similar loss for position 17-20, 25, and 29 in the C-terminus (Wisniewski et al. 2016). Consistent with this, the C-terminal of GLP-2 orientates towards a hydrophilic cavity in the NMR structure (Venneti et al. 2011). Thus, the N-terminal part of GLP-2 plays a central role in receptor activation, while the C-terminus adopts an alpha-helical conformation that plays a central role of receptor binding of GLP-2 consistent with the suggested “two-step” activation model of class B1 GPCRs, a model that is now much more refined (Liang et al. 2018; Qiao et al. 2020; Wu et al. 2020; Zhang et al. 2018; Zhang et al. 2017; Zhao et al. 2020).
The M10Y-modification barely changed the functional properties of the two endogenous hGLP-2 variants, demonstrating, in agreement with the model discussed above, that the Met10 of GLP-2 neither plays an important role in ligand binding nor receptor activation. According to the NMR structure, Met10 is positioned at the beginning of the alpha-helix and is not part of the binding interface of the GLP-2R (Venneti et al. 2011). Consistent with this, Wisniewski et. al. replaced the oxidation and alkylation-prone Met residue at position 10 of hGLP-2 by the isosteric Nor-leucine (Nle) (Wisniewski et al. 2016). Met is characterized by a sulfur atom in the sidechain, which is highly sensitive to reactive oxygen species (ROS) that often changes structural and functional properties of proteins (Black et al. 1991; Kim et al. 2014). ROS-mediated oxidation occurs by the addition of a single oxygen molecule to the sulfur atom, forming methionine sulfoxide (MetSO) (Kim et al. 2014), which creates a chiral center around the sulfur atom and overall results in a stiffer and more polar side chain compared to the unoxidized Met residue (Black et al. 1991). These changes can have profound structural and functional consequences (Chao et al. 1997; Hoshi et al. 2001; Gu et al. 2015; Sugamura et al. 2011). To protect for oxidative damage of the Met in GLP-2 during the oxidative iodination, and since Met is dispensable for GLP-2 function (Drucker et al. 2013; Venneti et al. 2011; Wisniewski et al. 2016; Yamazaki et al. 2013), we replaced Met10 with a Tyr residue. Thereby we created a target site for oxidative iodination using [125I] in the full agonist (GLP-2(1-33)) and in the antagonist and partial agonist (GLP-2(3-33)). These modifications created the two peptides; hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y). These two M10Y-substituted peptides acted as their wildtype counterparts, and with these, we were in a unique position allowing us to investigate both agonist [I125]-hGLP-2(1-33,M10Y) and antagonist [I125]-hGLP-2(3-33,M10Y) binding.
The similar affinities (KD) support the main role of the N-terminus in receptor activation and not in receptor binding (Couvineau et al. 2011). Moreover, the higher Bmax for the antagonist follows the general trend for more antagonist binding conformations versus agonist conformations of GPCRs ( Rosenkilde et al. 1994). Interestingly, for the first time among class B1 GPCRs, we describe the binding kinetics of a peptide agonist in comparison with a peptide antagonist and show, that the on-rate for the antagonist is significantly faster than for the agonist. Binding kinetics parameters, including kon and koff, have been highlighted to be more important in describing a ligand’s in vivo efficacy and the onset of action, than the classical parameters such as KD and KI (Velden et al. 2020). The slower on-rate for the agonist could reflect a more complex binding compared to the antagonist in line with expected induction of active receptor states (Zhang et al. 2018). When comparing the apparent affinities for the agonists and the antagonists obtained in competition with two radioligands, we observed similar affinities irrespective of choice of radioligand. This suggests that all four ligands (initially) interact similarly with the ECDs of the hGLP-2R, and that the receptor easily interchanges between (sequential) conformations induced by the agonist and the antagonist.
The location of the GLP-2R remains controversial in both rodent and humans. It has been reported that the GLP-2R mRNA transcript and protein is expressed in SEMFs (El-Jamal et al. 2014; Ørskov et al. 2005). Here we confirm receptor expression at the protein level in SEMFs in the intestine and in the pancreatic islet cells of mice by using the agonistic radioligand [I125]-hGLP-2(1-33,M10Y). The prevention of [125I]-hGLP-2(1-33,M10Y) labeling by co-injection with excess amounts of unlabeled hGLP-2(1-33,M10Y) demonstrates the specificity of [125I]-hGLP-2(1-33,M10Y) binding. The strong staining of the pancreatic islet cells by [125I]-hGLP-2(1-33,M10Y) could result from GLP-2R expression in the pancreatic islet cells, in agreement with what was previously shown at the mRNA level (De Heer et al. 2007). Alternatively, it could result from cross-interaction of GLP-2 with the GLP-1R, or a combination of the two. The strong binding properties of both radioligands to the mGLP-1R, and the low potency activation of the hGLP-1R by GLP-2(1-33) and GLP-2(1-33, M10Y), demonstrate that the pancreatic staining could be a result of GLP-1R binding. Also, promiscuity is known within GPCRs, demonstrated by the activation of the GIPR by GLP-2 (Skov-Jeppesen et al. 2019), the binding and activation of both the GLP-1R and the GCGR by oxyntomodulin (Holst et al. 2018; Jorgensen et al. 2007), and the activation of the mGLP-1R by glucagon (Svendsen et al. 2018). Thus, cross-activation is a common phenomenon within class B1 GPCRs, which is reflected in the high sequence similarities observed among the receptors and across species. For rodent GLP-2R’s, 81% and 79% sequence identities are found for the mGLP-2R and rGLP-2R, respectively, explaining the high-affinity binding observed for both radioligands to the rodent GLP-2R’s. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬
As we observed no binding of either hGLP-2 radioligand to the rGLP-2R, future autoradiography studies in rats would eliminate the binding of GLP-2 to the GLP-1R. Another possibility would be to use GLP-1R knock-out (KO)-mice, or eliminate hGLP-1R binding by modifications of GLP-2 at the C-terminus, as suggested recently in study where replacement of position 11 and/or 16 of hGLP-2(1-30) eliminated hGLP-1R actively, while retaining high hGLP-2R activity (Wisniewski et al. 2016). Thus, it is possible to decrease GLP-2 binding to the GLP-1R without compromising the GLP-2R binding.