Introduction. Amylin is a 37-amino acid peptide hormone involved in metabolism, glucoregulation and satiety and consequently has been investigated as a therapy for diabetes and obesity. Amylin is a Family B G protein-coupled receptor (GPCR) peptide ligand, proposed to activate its receptors via a two-domain model of binding whereby the peptide N-terminus is important for receptor activation. The calcitonin family, of which amylin is a member, all possess a highly conserved N-terminal disulphide loop which is critically important for receptor activation. Nevertheless, very little is currently known about the significance of the individual N-terminal residues of amylin in receptor activation.
Aims. To investigate the role of the N-terminal residues of human amylin in receptor activation by completing an alanine scan of the first 17 amino acids. Native alanine residues were substituted with glycine.
Methods. Fmoc solid-phase peptide synthesis was used to make peptide analogues, followed by cleavage, precipitation, oxidation and purification of the peptides. All were amidated and contained the disulphide bond. Biological functional assays were carried out in transfected COS7 cells expressing calcitonin or amylin receptors. Cyclic AMP production upon peptide stimulation was measured as an indicator of receptor activation.
Results. Many of the N-terminal alanine analogues caused minor to moderate reductions in potency. A key residue was threonine at position 6 within the disulphide loop; when replaced with alanine significant reductions in Emax resulted. Surprisingly, other loop analogues did not significantly alter human amylin activity, suggesting that residues beyond the loop play an important role in receptor activation.
Discussion. N-terminal residues which encompass the peptides “activation loop” have disproportionate and differential impacts on receptor activation. The strongly conserved threonine at position 6 is clearly a key residue in the N-terminal loop.