INTRODUCTION
Glucagon-like peptide-2 (GLP-2) is a gut hormone consisting of 33 amino acids (GLP-2(1-33)), which is a product of the pro-glucagon gene secreted from the enteroendocrine L-cells of the small intestine upon nutrient ingestion. GLP-2(1-33) is cleaved by the ubiquitous protease dipeptidyl peptidase-4 (DPP-4), resulting in the degradation product GLP-2(3-33) (Hartmann et al. 2000; Holst et al. 2000). In mice, administration of GLP-2(1-33) promotes growth of the small and large intestine (Drucker et al. 1996; Thulesen et al. 2002), stimulates proliferation of the crypt cell, nutrient absorption as well as promoting healing and maintenance of epithelial integrity (Drucker, 2013; Dubé, 2006). These intestinotrophic actions of GLP-2 have been exploited therapeutically with the use of the DPP-4 resistant GLP-2(1-33) analogue Teduglutide, that since 2012 has been used in the treatment of short bowel syndrome (SBS) in adults (Jeppesen et al. 2001; Kim et al. 2017). In addition, a four month clinical study showed that GLP-2(1-33) has an anti-catabolic effect on the bone tissue by inhibiting bone resorption (as measured by the bone marker CTX (C-terminal telopeptide)), as shown by our group and others (Askov-Hansen et al. 2013; Gottschalck et al. 2008; Gottschalck et al. 2008; Henriksen et al. 2009; Henriksen et al. 2003; Henriksen et al. 2007).
The metabolite GLP-2(3-33) acts as a partial agonist of the GLP-2 receptor (GLP-2R) (EC50 of ~6 nM and Emax of ~15% of GLP-2(1-33)) with competitive antagonistic properties on the human (hGLP-2R) in vitro andin vivo (Thulesen et al. 2002). Structurally, GLP-2 is closely related to the peptide hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GIP (secreted from enteroendocrine K-cells) and GLP-1 (co-secreted with GLP-2 from L-cells) are important insulinotropic hormones, whereas GLP-2 is inactive in this respect (Schiellerup et al. 2019). GLP-1 analogs are widely used as treatment for type 2 diabetes mellitus (T2DM) and obesity, and more recently, a dual-agonist of GLP-1 and GIP showed promising effects within this field (Coskun et al. 2018; Willard et al. 2020)
The GLP-2R is a G protein-coupled receptor (GPCR), belonging to the subclass B1 of the GPCR family, which comprises of 15 receptors including the GLP-1 receptor (GLP-1R), the GIP receptor (GIPR), the glucagon receptor (GCGR), the Secretin receptor (SecretinR), and the vasoactive intestinal peptide 1 and 2 receptors (VPAC1R and -2R) (Culhane et al. 2015; Gabe et al. 2020; Kenakin et al. 2010). High resolution structures of class B1 GPCRs (but not yet GLP-2R) combined with mutation studies, have enabled the analysis of the active, intermediate and inactive conformations of the receptor, thereby revealing residues that are essential for ligand binding and/or activation (Gabe et al. 2020; Parthier et al. 2009; Song et al. 2017; Zhang et al. 2018; Zhang et al. 2017). Currently, the leading paradigm regarding activation of class B1 GPCRs is the “two-step” mechanism, suggesting that the peptide ligand switches between an overall disordered and more ordered alpha-helical secondary structure, where the N-terminal part of the extracellular domain (ECD) of the receptor recognizes the C-terminal or middle part of the ligand for interaction, where after the N-terminal part of the ligand orientates towards and docks into the transmembrane domains (TMDs) of the receptor (Gabe et al. 2020; Parthier et al. 2007). The two-step mechanism is a simplified model of a complex network of conformational changes that takes place upon activation (Liang et al. 2018; Liang et al. 2018; Sasaki et al. 1975; Schwartz et. al. 2017; Venneti et al. 2011; Zhang et al. 2017; Zhao et al. 2019). Signaling through class B1 receptors, including the GLP-2R, mainly occurs through Gαs-coupling, thereby evoking multiple signaling cascades, including increased levels of the downstream second messenger cyclic adenosine monophosphate (cAMP) (Correll et al. 2014). By immunofluorescence microscopy, Estall et al. showed that the C-terminus of the GLP-2R recruits β-arrestin-2 following agonist stimulation, but that the recruitment is not required for Gαs-coupling, desensitization, and receptor endocytosis of GLP-2R (Estall et al. 2005). Functional consequences of β-arrestin recruitment by the GLP-2R have not yet been described, although important effects of recruitments have been demonstrated for other class B1 GPCRs, including the GIPR (Gabe et al. 2018) and the GLP-1R (Roussel et al. 2016; Whalen et al. 2011).
Although the GLP-2R was cloned for the first time in 1999, the precise tissue and cellular localization of GLP-2R expression remains controversial, undoubtedly due to the lack of specific antibodies. Messenger RNA (mRNA) transcripts of the GLP-2R are found within gastro-intestinal tissues (stomach, duodenum, jejunum, ileum, colon and intestinal ganglion cells) of various species, including human and rodents (Bjerknes et al. 2002; El-Jamal et al. 2014; Guan et al. 2006; Munroe et al. 1999; Ørskov et al. 2005; Pedersen et al. 2015; Yusta et al. 2000). El-Jamal N et. al. have demonstrated GLP-2R mRNA transcripts within the intestinal subepithelial myofibroblasts (SEMF) cell line, CCD-19Co (El-Jamal et al. 2014), supporting earlier studies (Ørskov et al. 2005), describing the expression of the GLP-2R protein throughout the small and large intestine, and particularly within the SEMFs of the GI tract by immunohistochemistry. Also, the mRNA transcript of the GLP-2R has been reported in extraintestinal tissues (fat, lymph nodes, bladder, spleen liver and hepatocytes) (El-Jamal et al. 2014; Yusta et al. 2000; Yusta et al. 2019). Interestingly, De Heer et. al. demonstrated the GLP-2R mRNA transcript in human and rat pancreas (De Heer et al. 2007), a tissue that is known for high expression levels of the GLP-1R (Richards et al. 2014). ‬‬‬‬‬‬‬‬‬Thus, there seems to be expression of the GLP-2R in several tissues.
Here, we present two new, high-affinity human GLP-2 (hGLP-2) radioligands with tyrosine (Tyr)-substitution at position 10 in the two naturally occurring GLP-2 peptides, the natural agonist GLP-2(1-33) and the antagonist and partial agonist GLP-2(3-33). With these, we determined active and in-active receptor conformations, the interchange between the two, and differential binding kinetics for the two radioligands. Furthermore, we performed autoradiography studies in mice to determined GLP-2R protein expression in vivo .