Partial Agonists and Inverse Agonists for β-Arrestin Translocation at the CGRP Receptor

DR Poyner1, P Harris2, R Kowalczyk2, M Brimble2, DL Hay3, GL Woodhall11School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK, 2School of Chemical Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1142, New Zealand, 3School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1142, New Zealand

Calcitonin gene-related peptide (CGRP) produces its effects at the CGRP receptor, a family B G protein-coupled receptor. The best characterised response to stimulation of this receptor is production of cAMP but it can couple to other pathways, including stimulation of β-arrestin translocation (1). In this study a variety of N-terminally modified CGRP peptides and SB273779 were examined on β-arrestin translocation. The N-terminus of CGRP is essential for receptor activation and SB273779 was previously described as an irreversible antagonist of CGRP-mediated cAMP production (2,3). The CGRP analogues were chosen to explore where derivatisation of the N-terminus would be possible.

Peptide CGRP analogues were synthesised on the solid phase using Fmoc chemistry at the University of Auckland and purified to >95% purity by HPLC. The structures were confirmed by electrospray mass spectroscopy. SB273779 was purchased from Tocris. Concentration-response curves were constructed using a Discoverx “Pathhunter eXpress β-arrestin” kit (93-0446E), with CHO-K1 cells expressing the human CGRP receptor and β-arrestin 2. The curves were analysed using Graphpad Prism 5 (logistic equation, fixed Hill slope) to obtain basal, Emax and pEC50 values. Emax’s were compared to 100% by Student’s t-test: EC50’s were compared to hαCGRP by Dunnett’s test after one-way ANOVA.

Table 1 illustrates that all the CGRP analogues were able to stimulate β-arrestin translocation, but they were all less potent than CGRP apart from [Ala8]-CGRP. With the exception of [Cys3]-CGRP, the analogues were all partial agonists. SB273779 was an inverse agonist with a pIC50 of 7.16 ± 0.08; it decreased the basal β-arrestin translocation to 27 ± 3% of the basal (n=3) (P<0.05 compared to 100%, Student’s t-test).

Table 1 pEC50 and Emax values for stimulation of β-arrestin translocation

Values are means ± sems of 3 determinations. *, P<0.05, compared to CGRP. h= human

This is the first report of the pharmacology of β-arrestin translocation at the CGRP receptor. It indicates that modification of the N-terminus of CGRP is likely to reduce efficacy and also shows that SB273779 is an inverse agonist at this response. Inverse agonist activity had not previously been reported for this agent.

References

1. Walker, C. S., Conner, A. C., Poyner, D. R., and Hay, D. L. (2010) Trends Pharmacol Sci 31, 476-483

2. Aiyar, N., Daines, R. A., Disa, J., Chambers, P. A., Sauermelch, C. F., Quiniou, M., Khandoudi, N., Gout, B., Douglas, S. A., and Willette, R. N. (2001) J Pharmacol Exp Ther 296, 768-775

3. Dennis, T., Fournier, A., St Pierre, S., and Quirion, R. (1989) J Pharmacol Exp Ther 251, 718-725