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 AM 22-52 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 CGRP 1 and AM2 receptors and AM could activate both AM 1 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. Copyright © 2004 Humana Press Inc. All rights of any nature whatsoever reserved.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors:a comparison of effects of adrenomedullin 22-52, CGRP 8-37 and BIBN4096BS

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 22-52 and CGRP 8-37) 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. 2. AM 22-52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA 2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat2), 7.00±0.05 (L6), 6.25±0.17(rCL/hRAMP2)). CGRP 8-37 (10 μM) resembled AM 22-52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent PA 2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0. 12 (L6)). 3. On CL/RAMP3 receptors, 10 μM CGRP 8-37 was an effective antagonist at all combinations (apparent pA 2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/ hRAMP3)). However, 10 μm AM 22-52 only antagonised AM at the hCL/hRAMP3 receptor (apparent PA 2 6.73±0.14). 4. BIBN4096BS (10 μM) 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 AM∼βCGRP>αCGRP. 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.

Interaction of calcitonin-gene-related peptide with its receptors

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28-37 and 8-18 are normally required for high-affinity binding, while residues 19-27 form a hinge region. The 28-37 region is almost certainly in direct contact with the receptor; 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of 28-37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.

A comparison of the actions of BIBN4096BS and CGRP 8-37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells

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 significantly 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, significantly less than 1. 4. On human Colony (Col) 29 cells, CGRP 8-37 had a significantly 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 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. 5. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. 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 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP 8-37.

Structure-function analysis of RAMP1 by Alanine Mutagenesis

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Structure-function analysis of RAMP1-RAMP3 chimeras

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

Mediators of monocyte activity in inflammation

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. CRP is currently one of the best markers of inflammatory disease and disease activity. One of the keys cells involved in inflammation within chronic inflammatory diseases is the monocyte. Monocytes are able to modulate inflammation through cytokine expression, cytosolic peroxide formation, adhesion molecule expression and subsequent adhesion/migration to sites of inflammation. CRP has been previously shown to bind directly to monocytes through Fc receptors. However this observation is not conclusive and requires further investigation. The effects of incubation of CRP with human primary and monocytic cell lines were examined using monocytic cytokine expression, adhesion molecule expression and adhesion to endothelial cells and intracellular peroxide formation, as end points. Monocytic intracellular signalling events were investigated after interaction of CRP with specific CRP receptors on monocytes. These initial signalling events were examined for their role in modulating monocytic adhesion molecule and cytokine expression. Monocyte recruitment and retention in the vasculature is also influenced by oxidative stress. Therefore the effect of 6 weeks of antioxidant intervention in vivo was examined on monocytic adhesion molecule expression, adhesion to endothelial cells ex vivo and on serum CRP concentrations, pre- and post- supplementation with the antioxidants vitamin C and vitaInin E. In summary, CRP is able to bind FcγRIIa. CRP binding FcγR initiates an intracellular signalling cascade that phosphorylates the non-receptor tyrosine kinase, Syk, associated with intracellular tyrosine activating motifs on the cytoplasmic tail of Fcγ receptors. CRP incubations increased phosphatidyl inositol turnover and Syk phosphorylation ultimately lead to Ca2+ mobilisation in monocytes. CRP mediated Syk phosphorylation in monocytes leads to an increase in CD 11b and IL-6 expression. CRP engagement with monocytes also leads to an increase in peroxide production, which can be inhibited in vitro using the antioxidants α-tocopherol and ascorbic acid. CRP mediated CD 11b expression is not redox regulated by CRP mediated changes in cytosolic peroxides. The FcyRIla polymorphism at codon 131 effects the phenotypic driven changes described in monocytes by CRP, where R/R allotypes have a greater increase in CD11b, in response to CRP, which may be involved in promoting the monocytic inflammatory response. CRP leads to an increase in the expression of pro-inflammatory cytokines, which alters the immune phenotype of circulating monocytes. Vitamin C supplementation reduced monocytic adhesion to endothelial cells, but had no effect on serum levels of CRP. Where long-term antioxidant intervention may provide benefit from the risk of developing vascular inflammatory disease, by reducing monocytic adhesion to the vasculature. In conclusion CRP appears to be much more than just a marker of ongoing inflammation or associated inflammatory disease and disease activity. This data suggests that at pathophysiological concentrations, CRP may be able to directly modulate inflammation through interacting with monocytes and thereby alter the inflammatory response associated with vascular inflammatory diseases.

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.

RAMPs and CGRP receptors

Receptor activity modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor (CLR) to produce the receptor for calcitonin gene-related peptide (CGRP). RAMP1 has two main roles. It facilitates the cell-surface expression of CLR. It is also essential for the binding of CGRP to the receptor. It seems likely that Y66, F93, H97 and F101, amongst other residues, form a binding site for CLR. These cluster together on the same face of the extracellular portion of RAMP1, probably close to where it enters the plasma membrane. Residues at the other end of RAMP1 are most likely to be involved in CGRP recognition, although it is currently unclear how they do this. Within this area, W74 is important for the binding of the nonpeptide antagonist, BIBN4096BS, although it does not seem to be involved in the binding of CGRP itself. It has been shown that there is an epitope within residues 23-60 of CLR that are essential for RAMP recognition. Under some circumstances, changes in the expression of RAMP1 can alter the sensitivity of cells to CGRP, demonstrating that regulation of its levels may be of physiological or pathophysiological importance.

RAMPs as drug targets

The receptor activity-modifying protein (RAMP) family of membrane proteins regulates G protein-coupled receptor (GPCR) function in several ways. RAMPs can alter their pharmacology and signalling as well as the trafficking of these receptors to and from the cell surface. Accordingly, RAMPs may be exploited as drug targets, offering new opportunities for regulating the function of therapeutically relevant RAMP-interacting GPCRs. For example, several small molecule antagonists of RAMP1/ calcitonin receptor-like receptor complexes, which block the actions of the neuropeptide calcitonin gene-related peptide are in development for the treatment of migraine headache.

The pharmacology of adrenomedullin 2/intermedin

Adrenomedullin 2 (AM2) or intermedin is a member of the calcitonin gene-related peptide (CGRP)/calcitonin family of peptides and was discovered in 2004. Unlike other members of this family, no unique receptor has yet been identified for it. It is extensively distributed throughout the body. It causes hypotension when given peripherally, but when given into the CNS, it increases blood pressure and causes sympathetic activation. It also increases prolactin release, is anti-diuretic and natriuretic and reduces food intake. Whilst its effects resemble those of AM, it is frequently more potent. Some characterization of AM2 has been done on molecularly defined receptors; the existing data suggest that it preferentially activates the AM receptor formed from calcitonin receptor-like receptor and receptor activity modifying protein 3. On this complex, its potency is generally equivalent to that of AM. There is no known receptor-activity where it is more potent than AM. In tissues and in animals it is frequently antagonised by CGRP and AM antagonists; however, situations exist in which an AM2 response is maintained even in the presence of supramaximal concentrations of these antagonists. Thus, there is a partial mismatch between the pharmacology seen in tissues and that on cloned receptors. The only AM2 antagonists are peptide fragments, and these have limited selectivity. It remains unclear as to whether novel AM2 receptors exist or whether the mismatch in pharmacology can be explained by factors such as metabolism. © 2011 The British Pharmacological Society.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance.

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution.