1 Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM ) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. 2 AM 22 -52 (10 mm) antagonised AM at all CL/RAMP2 complexes (apparent pA 2 values: 7.3470.14 (hCL/hRAMP2), 7.2870.06 (Rat 2), 7.0070.05 (L6), 6.2570.17 (rCL/hRAMP2)). CGRP 8 -37 (10 mm) resembled AM 22 -52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA 2 values: 7.0470.13 (hCL/hRAMP2), 6.7270.06 (Rat2), 7.0370.12 (L6)). 3 On CL/RAMP3 receptors, 10 mm CGRP 8 -37 was an effective antagonist at all combinations (apparent pA 2 values: 6.9670.08 (hCL/hRAMP3), 6.1870.18 (rCL/rRAMP3), 6.4870.20 (rCL/ hRAMP3)). However, 10 mm AM 22 -52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA 2 6.7370.14). 4 BIBN4096BS (10 mm) did not antagonise AM at any of the receptors. 5 Where investigated (all-rat and rat/human combinations), the agonist potency order on the CL/ RAMP3 receptor was AMBbCGRP4aCGRP. 6 rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. 7 This study shows that on CL/RAMP complexes, AM 22 -52 has significant selectivity for the CL/ RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP 8 -37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM22-52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for betaCGRP. Here, CGRP8-37 is either equipotent or more effective as an antagonist than AM22-52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that betaCGRP might be able to activate both CGRP1 and AM2 receptors and AM could activate both AM1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2.

1 Structure-activity relationships for the binding of human a-calcitonin gene-related peptide 8 ± 37 (haCGRP 8 ± 37 ) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (haCGRP stimulation of adenylate cyclase). ]-CGRP 8 ± 37 , where the amphipathic nature of the N-terminal a-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than haCGRP 8 ± 37 . 5 On SK-N-MC cells, haCGRP 8 ± 18 , 28 ± 37 (M433) and mastoparan-haCGRP 28 ± 37 (M432) had apparent pKBs of 6.64+0.16 and 6.42+0.26, suggesting that residues 19 ± 27 play a minor role in binding. The physico-chemical properties of residues 8 ± 18 may be more important than any speci®c side-chain interactions. 6 M433 was almost as potent as haCGRP 8 ± 37 on Col 29 cells (apparent pKB=6.17+0.20). Other antagonists were inactive.

No abstract

1 The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. 2 On human SK-N-MC cells (expressing human calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA 2 of 9.95 although the slope of the Schild plot (1.37+0.16) was signi®cantly greater than 1. 3 On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA 2 of 9.25 and a Schild slope of 0.89+0.05, signi®cantly less than 1. 4 On human Colony (Col) 29 cells, CGRP 8 ± 37 had a signi®cantly lower pA 2 than on SK-N-MC cells (7.34+0.19 (n=7) compared to 8.35+0.18, (n=6)). BIBN4096BS had a pA 2 of 9.98 and a Schild plot slope of 0.86+0.19 that was not signi®cantly di erent from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK-N-MC cells. 5 On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 mM. CGRP 8 ± 37 had a pA 2 of 6.72 against adrenomedullin. 6 BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK-N-MC cells and the CGRP-responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent`non-competive' behaviour. At concentrations of up to 10 mM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP 8 ± 37 .