Stephen G. Howitt

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS

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 (AM22–52 and CGRP8–37) 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. AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)). On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14). BIBN4096BS (10 μM) did not antagonise AM at any of the receptors. Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

The pharmacology of adrenomedullin receptors and their relationship to CGRP 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 βCGRP. 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 βCGRP 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.

A comparison of the actions of BIBN4096BS and CGRP8–37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells

The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. On human SK‐N‐MC cells (expressing human calcitonin receptor‐like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1. On human Colony (Col) 29 cells, CGRP8–37 had a significantly lower pA2 than on SK‐N‐MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA2 of 9.98 and a Schild plot slope of 0.86±0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK‐N‐MC cells. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP8–37 had a pA2 of 6.72 against adrenomedullin. 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 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP8–37.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

Structure‐activity relationships for the binding of human α‐calcitonin gene‐related peptide 8–37 (hαCGRP8–37) 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 (hαCGRP stimulation of adenylate cyclase). On SK‐N‐MC cells the potency order was hαCGRP8–37>hαCGRP19–37=AC187>rat amylin8–37> hα[Tyr0]‐CGRP28–37 (apparent pKBs of 7.49±0.25, 5.89±0.20, 6.18±0.19, 5.85±0.19 and 5.25±0.07). The SK‐N‐MC receptor appeared CGRP1‐like. On Col 29 cells, only hαCGRP8–37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB=6.48±0.28). Its receptor appeared CGRP2‐like. hα[Ala11,18]‐CGRP8–37, where the amphipathic nature of the N‐terminal α‐helix has been reduced, bound to SK‐N‐MC cells a 100 fold less strongly than hαCGRP8–37. On SK‐N‐MC cells, hαCGRP8–18, 28–37 (M433) and mastoparan‐hαCGRP28–37 (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 specific side‐chain interactions. M433 was almost as potent as hαCGRP8–37 on Col 29 cells (apparent pKB=6.17±0.20). Other antagonists were inactive.

Heterodimers and family-B GPCRs: RAMPs, CGRP and adrenomedullin

RAMPs (receptor activity-modifying proteins) are single-pass transmembrane proteins that associate with certain family-B GPCRs (G-protein-coupled receptors). Specifically for the CT (calcitonin) receptor-like receptor and the CT receptor, this results in profound changes in ligand binding and receptor pharmacology, allowing the generation of six distinct receptors with preferences for CGRP (CT gene-related peptide), adrenomedullin, amylin and CT. There are three RAMPs: RAMP1-RAMP3. The N-terminus appears to be the main determinant of receptor pharmacology, whereas the transmembrane domain contributes to association of the RAMP with the GPCR. The N-terminus of all members of the RAMP family probably contains two disulphide bonds; a potential third disulphide is found in RAMP1 and RAMP3. The N-terminus appears to be in close proximity to the ligand and plays a key role in its binding, either directly or indirectly. BIBN4096BS, a CGRP antagonist, targets RAMP1 and this gives the compound very high selectivity for the human CGRP(1) receptor.

The pharmacology of CGRP-responsive receptors in cultured and transfected cells

Historically, CGRP receptors have been classified as CGRP(1) or CGRP(2) subtypes, chiefly depending on their affinity for the antagonist CGRP(8-37). It has been shown that the complex between calcitonin receptor-like receptor (CRLR or CL) and receptor activity modifying protein (RAMP) 1 provides a molecular correlate for the CGRP(1) receptor; however, this does not explain the range of affinities seen for CGRP(8-37) in isolated tissues. It is suggested that these may largely be explained by a combination of methodological factors and CGRP-responsive receptors generated by CL and RAMP2 or RAMP3 and complexes of RAMPs with the calcitonin receptor.

The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general.

The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions.

The role of individual residues in the 8‐18 helix of CGRP8‐37 in promoting high‐affinity binding to CGRP1 receptors expressed on rat L6 and human SK‐N‐MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP‐mediated increase in cyclic AMP production and the binding of [125I]‐human αCGRP. Arg11 and Arg18 were replaced by serines to give [Ser11,18]CGRP8‐37. These bound with pKi values <6 to SK‐N‐MC cells and had apparent pA2 values of 5.81±0.04 and 5.31±0.11 on SK‐N‐MC and L6 cells. CGRP8‐37 had a pKi of 8.22 on SK‐N‐MC cells and pKb values on the above cell lines of 8.95±0.04 and 8.76±0.04. The arginines were replaced with glutamic acid residues. [Glu11]CGRP8‐37 had a pKb of 7.14±0.14 on SK‐N‐MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu18]CGRP8‐37 had a pKb of 7.10±0.0.08 on SK‐N‐MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. Leu12, Leu15 and Leu16 were replaced by benzoyl‐phenylalanine (bpa) residues. On SK‐N‐MC cells, the apparent pA2 values of [bpa12]‐, [bpa15]‐ and [bpa16]CGRP8‐37 were respectively 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and <6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n=3). It is concluded that the Arg11 and Arg18 are involved in specific electrostatic interactions with other residues, either on the CGRP1 receptors or elsewhere on CGRP8‐37. Leu16 is in a conformationally restricted site when CGRP8‐37 binds to CGRP1 receptors, unlike Leu12 and Leu15. 

The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

Potency orders were determined for a series of agonists and antagonists on the calcitonin gene‐related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. AC187 had a pIC50 of 6.8±0.10, making it 14 fold less potent as an antagonist than CGRP8–37 (pIC50, 7.95±0.14). Amyline8–37 was equipotent to AC187 (pIC50, 6.6±0.16) and CGRP19–37 was 3 fold less potent than either (pIC50, 6.1±0.24). [Ala11]‐CGRP8–37 was 6 fold less potent than CGRP8–37, (pIC50, 7.13±0.14), whereas [Ala18]‐CGRP8–37 was approximately equipotent to CGRP8–37 (pIC50, 7.52±0.15). However, [Ala11,Ala18]‐CGRP8–37 was over 300 fold less potent than CGRP8–37 (pIC50, 5.30±0.04). [Tyr0]‐CGRP28–37, amylin19–37 and adrenomedullin22–52 were inactive as antagonists at concentrations of up to 1 μM. Biotinyl‐human α‐CGRP was 150 fold less potent than human α‐CGRP itself (EC50 values of 48±17 nM and 0.31±0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)2,7]‐CGRP was inactive as an agonist. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8–37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.