The role of position 6 of calcitonin gene-related peptide in receptor binding and activation

Paul Harris1, Renata Kowalczyk1, James Barwell2, Margaret Brimble1, Alex Conner3, Debbie Hay4, David Poyner2. 1School of Chemical Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, 1142, New Zealand, 2School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK, 3Medical School, University of Warwick, Coventry, CV4 7AL, UK, 4School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, 1142, New Zealand

The threonine at position 6 of CGRP is absolutely conserved in the equivalent position of all members of the CGRP family of peptides. Previously we reported that substitution of this position by cysteine converted the analogue to an antagonist (Barwell et al., 2010). In this study, we have explored the structural requirements at this position by substituting it with alanine, aspartate, serine and lysine in human alpha CGRP.

Peptides 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. The activity of the peptides was assayed on SK-N-MC cells which express an endogenous CGRP receptor, measuring the pEC50 for cAMP production as described previously (Poyner et al., 1998), using Graphpad Prism 4.00 for data-fitting. Where antagonist activity was being assessed, the cells were pretreated with peptides for 10 minutes prior to the addition of CGRP. pEC50 values were compared to that of human alpha CGRP using Dunnett’s test following one-way ANOVA. To assess if Emax values differed from wild-type (100), 95% confidence limits were calculated using Student’s t statistic.

CGRP stimulated cAMP production with a pEC50 of 8.48±0.10 (n = 4). For Ser-6 and Ala-6 CGRP, the EC50s were both significantly reduced (7.27±0.26 and 6.59±0.20, P < 0.001), but the Emax values were unchanged (100±10 and 72±12), all n = 4. At concentrations of up to 10 μM, neither Lys-6 nor Asp-6 CGRP caused any stimulation of cAMP production (n = 4). However, both analogues caused small but significant rightward shifts in the concentration response curve for CGRP (pEC50 for CGRP alone, 8.66±0.19; with 10μM Asp-6 CGRP, 8.04±0.04; with 10μM Lys-6 CGRP, 8.11±0.09, P<0.05, n = 4). These values are consistent with the two analogues having pA2 values of ∼5.5.

These data confirm the importance of a threonine at position 6 of human alpha CGRP. Even replacement by serine reduces potency; in addition to replacement by cysteine, replacement by either aspartate or lysine also removes measurable efficacy when assayed by cAMP production in SK-N-MC cells. It is possible that Thr-6 of CGRP takes part in a hydrogen bond with a donor/acceptor on calcitonin receptor-like receptor (a subunit of the CGRP receptor) in a highly sterically constrained environment.

Barwell, J. et al., (2010), http://www.pA2online.org/abstracts/Vol8Issue1abst115P.pdf

Poyner, D.R. et al., (1998), Br. J. Pharmacol., 124, 1659-1666.

This work was supported by ARCHA, Aston University.