Characterising the signalling profiles of the calcitonin and amylin receptors

Abstract

Amylin is a peptide hormone released from the pancreas in response to food intake. This results in meal ending satiation and a lowering of blood glucose levels. Because of these properties amylin receptors (AMY) offer therapeutically relevant targets for the treatment of diabetes and obesity. Pramlintide, an amylin analogue, is currently approved for the treatment of diabetes. Amylin and pramlintide act through one of the three amylin receptors, formed by the association of the calcitonin receptor (CTR) and one of three receptor activity modifying proteins (RAMPs). Very little is known about amylin receptor signalling or which amylin receptor is therapeutically relevant. Understanding how amylin receptors signal may allow for better targeted drug design. The present study aimed to characterise calcitonin and amylin receptor signalling and analysed any potential signalling bias. Cos-7 cells were transfected with CTR alone or with the relevant RAMP. Signalling responses to the peptides calcitonin, amylin, αCGRP and pramlintide were measured using functional assays for the activation of cAMP, IP1, ERK1/2 and CREB signalling. The pharmacological properties of specific N-terminally alanine modified amylin and pramlintide peptides were investigated to determine the role these residues play in signalling. Peptide and RAMP dependent differences in signalling were observed at the calcitonin and amylin receptors. Additionally, biased signalling was observed for the CTR, AMY2 and AMY3 receptors. Modification of human amylin at position 6 had reduced activity for a number of pathways while other modifications displayed similar signalling profiles to wild-type human amylin and pramlintide. Single amino acid modifications appeared to be sufficient to induce signalling bias at the calcitonin and amylin receptors. The differences in signalling and pharmacology at each amylin receptor indicate possible RAMP dependent biased agonism. RAMPs may contribute to bias by contributing to the peptide binding pocket, intracellular signalling via the C-terminus or modulating/constraining the CTR conformation. Additionally, interesting pharmacological properties were also observed for ERK1/2 and CREB phosphorylation in response to N-terminal modified hAmy and pramlintide peptides. These findings will provide insight for the design of future amylin mimetics which act as biased agonists to selectively activate beneficial signalling events.