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    • With the exception of R none of the other residues

    2022-01-19

    With the exception of R5.40310, none of the other residues would appear to be in a position to mediate direct agonist interactions in the active state GLP-1R structures. ECL2 within the GLP-1R is important for affinity and efficacy of both extended and processed GLP-1 peptides [24]. Though distal from the peptide binding site, R3.30227 and K4.64288 form interactions with the top of TM4 and residues in ECL2, respectively, in the active receptor conformation and may act to stabilize this loop for peptide engagement. Therefore, mutations of these key stabilising interactions would likely result in disrupted or altered interactions of residues within ECL2 with peptide ligands. Mutation of R3.30227 resulted in a very similar profile to R5.40310 with large reductions in affinity of truncated peptides and no change in affinity, but a large reduction in cAMP efficacy for extended peptides (Fig. 9). Similarly, K4.64288 is crucial for peptide affinity for all four GLP-1 variants, but with a much larger role in the binding of the processed peptides. However, in contrast, this residue plays a similar, important role in propagation of signaling (for cAMP, pERK1/2 and calcium mobilization) for all peptides. Taken together, these data support a different binding and activation mode of extended and processed peptides. This was further supported by the mutations of R2.60190 and H6.52363 in the central polar network that reside just below the peptide binding pocket. These mutant residues caused a >15 fold reduction in affinity for GLP-1(7-36)NH2 or GLP-1(7-37) with no effect on the affinity of the extended peptides compared with wild type suggesting that these residues of the central polar network are important in maintaining the conformation of the receptor binding pocket that is required for high affinity interaction with the processed peptides. Alanine substitution of many of the conserved polar residues resulted in similar patterns of efficacy change for all peptides, which may reflect their universal role in mediating conformational transitions associated with activation (Fig. 9). However, as noted for other peptide agonists [27], [28], [29], TPMPA of some of these residues, such as H6.52363 and S2.56186, resulted in markedly diverse efficacy changes at the different pathways, supporting their role in the differential stabilization of receptor conformations responsible for alternate effector coupling (Fig. 9). Consistent with the processed and extended versions of the peptides having distinct interactions with the receptor, mutation of residues distal from the peptide binding site, towards the base of the receptor that opens up to engage with different transducer proteins, results in changes in efficacy that can be grouped into effects according to the length of the activating peptide. Thus, N7.61406A and Y3.53250A more strongly reduced ERK1/2 efficacy for GLP-1(7-36)NH2 or GLP-1(7-37) compared with the GLP-1(1-36)NH2 or GLP-1(1-37), whereas neither mutation had any effect on cAMP accumulation. Conversely, there are instances where a conserved polar mutation resulted in one of the 4 tested peptides becoming an outlier, such as R5.56326, whose mutation produces a large loss in GLP-1(7-37) cAMP efficacy not seen with the other peptides. We speculate that this may be related to changes in the dynamics of the extracellular domain interactions with the transmembrane bundle that have been shown to be important for GLP-1R mediated cAMP production [36]. Consequently, differences in peptide-N-terminal domain interaction produced by differences in the peptide carboxy-terminus could alter interactions between the N-terminal domain and the transmembrane bundle resulting in differential receptor engagement. Overall, we interpret our data to indicate that the processed and extended GLP-1 peptides interact quite differently with the receptor and this differential engagement and signal transduction is clearly revealed through the mutation of conserved polar residues in the receptor transmembrane bundle, as illustrated in Fig. 9. In addition, there appear to be subtle differences between glycine extended and amidated peptides in terms of receptor engagement and activation. This suggests there could well be hitherto unappreciated texture to GLP-1R physiology modulated through the secretion of these differentially processed GLP-1 peptides.