Differential internalisation of the CGRP and AMY1 receptor
Authors: Rees Tayla Anne , Gingell Joseph , Siow Andrew , Harris Paul , Brimble Margaret , Hay Debbie , Walker Christopher 
Published: April 2020

Calcitonin gene‐related peptide (CGRP) is a neuropeptide which plays a key role in migraine pathogenesis. CGRP is able to potently activate two G Protein‐coupled receptors (GPCRs), the canonical CGRP receptor which consists of calcitonin‐like receptor (CLR) in complex with receptor activity‐modifying protein 1 (RAMP1) and the AMY1 receptor, comprised of the calcitonin receptor (CTR) and RAMP1. The AMY1 receptor is also potently stimulated by amylin and amylin mimetics, including pramlintide. The CGRP and AMY1 receptor are expressed in several sites important for pain and migraine, such as the trigeminal ganglia. The CGRP receptor is known to internalise and signal from endosomes, however little is known about AMY1 receptor regulation. In this study we aimed to characterise and compare the internalisation of the CGRP and AMY1 receptor.

In order to study internalisation, we developed and characterised novel Cy5‐labelled hαCGRP and pramlintide. [Cy53]‐hαCGRP and [Cy521]‐pramlintide were selected for this study as they displayed similar cAMP signalling potencies to unlabelled peptides in Cos‐7 cells transfected with the CGRP or AMY1 receptor. 10 nM of [Cy53]‐hαCGRP or [Cy521]‐pramlintide was used to investigate receptor internalisation. In CGRP receptor transfected HEK293S cells, 10 nM [Cy53]‐hαCGRP formed intracellular spots which co‐localised with the CGRP receptor components (CLR and RAMP1) and the endosomal marker EEA1. However, at the AMY1 receptor 10 nM [Cy53]‐hαCGRP was primarily localised to cell surface with the AMY1 receptor components (CTR and RAMP1) and did not colocalise with EEA1. When quantified (spots/cell) the CGRP receptor had significantly greater internalisation of [Cy53]‐hαCGRP than the AMY1 receptor. 10 nM of [Cy521]‐pramlintide displayed little quantifiable internalisation at the AMY1 receptor. To confirm that the observed difference in internalisation was not concentration‐dependent, concentration responses for internalisation were determined. Internalisation of [Cy53]‐hαCGRP was significantly lower at the AMY1 receptor (spots/cell pEC50 7.96 ± 0.13, Emax 3.15 ± 0.36) than the CGRP receptor (spots/cell pEC50 8.80 ± 0.06, Emax 5.64 ± 0.19). To confirm this pattern of internalisation we measured the cell surface expression of receptor components using an ELISA in transiently transfected HEK293S and Cos‐7 cells. The CGRP receptor components (CLR and RAMP1) exhibited a significant reduction of cell surface expression in response to unmodified hαCGRP, while at the AMY1 receptor no significant internalisation was observed in response to hαCGRP or hAmylin.

The AMY1 receptor displays little internalisation in response to CGRP, pramlintide and amylin, while the CGRP receptor internalises robustly in response to CGRP, as expected. These data suggest that CGRP activity through the two receptors could be regulated by distinct internalisation profiles. This may have implications for physiology and drug treatment. This is the first report describing regulation of the AMY1 receptor.

Support or Funding Information

This research supported by Health Research Council of NZ and the Royal Society of NZ grants and University of Auckland Doctoral Scholarship (TR).