Structure Function Relationships in the Adrenomedullin N-terminus: An Alanine Scan of Residues 15-30

M Garelja, HA Watkins, P Harris, MA Brimble, G Williams, R Kowalczyk, DL Hay. The University of Auckland, Auckland, New Zealand

Human adrenomedullin (hAM) is a peptide with a wide range of biological effects. It has numerous roles, from vascular growth and regulation, to embryonic development (1). Although nominally 52 amino acids in length, the first 14 residues are not important for function (2). However the disulphide ring between residues 16-21 and surrounding residues are thought to be important for receptor activation.

To test this hypothesis, analogues of hAM15-52 were synthesised, incorporating individual alanine (Ala) substitutions from residues 15 to 30, omitting Cys16, Cys21, and Ala27. HEK293S cells were transiently transfected with the three pharmacologically relevant receptors (CGRP, AM1 and AM2 receptors). These cells were stimulated with hAM15-52 analogues and their ability to elicit a cAMP response was measured (3). Affinity was determined for all analogues apart from [Ala15]-hAM15-52 and [Ala19]-hAM15-52, using 125I-rAM in whole cells at the AM1 receptor.

Table 1. pEC50 values for analogues at the AM1 receptor. Values are mean ± SEM, with the number of experiments in parentheses. *P<0.05 , **P<0.01, ***P<0.001 by unpaired t-test against AM15-52.

Substitution of Gly15 or Thr22 for Ala resulted in <5-fold changes in potency at all receptors (AM1 receptor data shown in Table 1). Ala substitutions of Arg17, Val23, or His28 caused a small (5-20 fold) but statistically significant reduction in potency compared to hAM15-52 at all receptors. For Gln24 and Lys25, small (<10-fold) reductions were observed at both AM receptors. Substitution of Thr20, Leu26 or Ile30 for Ala reduced the maximal cAMP generated at all receptors, accompanied by large potency reductions. Substitution of Gly19 for Ala enhanced potency at the CGRP receptor (control pEC50: 7.85 ± 0.05 (4), [Ala19]-hAM15-52: pEC50, 8.93 ± 0.13 (4), P<0.001) but had no effect at the two AM receptors. There were no reductions in pIC50 values compared to AM15-52 in a radioligand binding assay.

This study has identified residues critical for receptor activation, creating a model in which important residues are found within the region of the disulphide ring of AM, and also residues up to position 30. It also leads to the hypothesis that Gly19 may be a key residue for determining pharmacological specificity between AM and CGRP receptors.

(1) Kuwasako K et al, Peptides 32:1540, 2011

(2) Santiago JA et al, Eur J Pharmacol 272:115, 1995

(3) Gingell JJ et al, Peptides 31:1400, 2010