Pharmacology of receptors for calcitonin gene-related peptide and amylin.

Calcitonin gene-related peptide (CGRP), a widespread neuropeptide with multiple actions, has substantial homology with amylin, a peptide implicated in insulin-resistant diabetes, and adrenomedullin, a recently discovered potent vasodilator. There is controversy over the existence of CGRP receptor subtypes, and whether independent receptors exist for amylin and adrenomedullin. In this article, the current status of CGRP receptor classification is reviewed by David Poyner, taking particular account of species differences, and evidence is presented supporting the existence of multiple receptors for CGRP, as well as independent binding sites for amylin.

Calcitonin gene-related peptide: multiple actions, multiple receptors.

Calcitonin gene-related peptide (CGRP) shows diversity both in its effects and its receptors. It is likely to have roles as a neurotransmitter, neuromodulator, local hormone and trophic factor. Its effects include rapid changes in neuronal activity, relaxation of many types of smooth muscle, actions on metabolism and changes in gene expression. Receptor heterogeneity has been revealed from experiments comparing agonist potency ratios and antagonist affinities. The evidence from these approaches is reviewed in this article and a speculative receptor classification scheme is proposed. Some of the likely future directions for CGRP research are discussed.

Soluble and membrane-bound muscarinic acetylcholine receptors.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

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

1. 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. 2. 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 CGRP19-32 was 3 fold less potent than either (pIC50, 6.1 +/- 0.24). 3. [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). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 microM. 5. Biotinyl-human alpha-CGRP was 150 fold less potent than human alpha-CGRP itself (EC50 values of 48 +/- 17 nM and 0.31 +/- 0.13 nM, respectively). At 1 microM, [Cys(acetomethoxy)2,7]-CGRP was inactive as an agonist. 6. 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.

Multiple receptors for calcitonin gene-related peptide and amylin on guinea-pig ileum and vas deferens.

1. The responses of the electrically stimulated guinea-pig ileum and vas deferens to human and rat calcitonin gene-related peptide (CGRP) and amylin were investigated. 2. The inhibition of contraction of the ileum produced by human alpha CGRP was antagonized by human alpha CGRP8-37 (apparent pA2 estimated at 7.15 +/- 0.23) > human alpha CGRP19-37 (apparent pA2 estimated as 6.67 +/- 0.33) > [Tyr0]-human alpha CGRP28-37. The amylin antagonist, AC187, was three fold less potent than CGRP8-37 in antagonizing human alpha CGRP. 3. Both human beta- and rat alpha CGRP inhibited contractions of the ileum, but this was less sensitive to inhibition by CGRP8-37 than the effect of human alpha CGRP. However, CGRP19-37 was twenty times more effective in inhibiting the response to rat alpha CGRP (apparent pA2 estimated as 8.0 +/- 0.1) compared to human alpha CGRP. 4. Rat amylin inhibited contractions in about 10% of ileal preparations; this effect was not antagonized by any CGRP fragment. Human amylin had no action on this preparation. 5. Both human and rat alpha CGRP inhibited electrically stimulated contractions of the vas deferens, which were not antagonized by 3 microM CGRP8-37 or 10 microM AC187. 6. Rat amylin inhibited the stimulated contractions of the vas deferens (EC50 = 77 +/- 9 nM); human amylin was less potent (EC50 = 213 +/- 22 nM). The response to rat amylin was antagonized by 10 microM CGRP8-37 (EC50 = 242 +/- 25 nM) and 10 microM AC187 (EC50 = 610 +/- 22 nM). 7. It is concluded that human alpha CGRP relaxes the guinea-pig ileum via CGRP1-like receptors, but that human beta CGRP and rat alpha CGRP may use additional receptors. These are distinct CGRP2-like and amylin receptors on guinea-pig vas deferens.

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 alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) 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 (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[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 halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (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 halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.

Modulation of the structure-binding relationships of antagonists for muscarinic acetylcholine receptor subtypes.

1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes.

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.

Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens.

1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD2s for CGRP, amylin and adrenomedullin of 7.90+/-0.11, 7.70+/-0.19 and 7.25+/-0.10 respectively). 3. CGRP8-37 (1 microM) and AC187 (10 microM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC50 of 8.91 and a capacity of 643 fmol mg(-1). Its selectivity profile was adrenomedullin> AC187>CGRP=amylin. It was clearly distinct from a site labelled by [125I]-CGRP (pIC50=8.73, capacity=114 fmol mg(-1), selectivity CGRP>amylin=AC187>adrenomedullin). [125I]-amylin bound to two sites with a total capacity of 882 fmol mg(-1). 6. Although CGRP has been shown to act at a CGRP2 receptor on the vas deferens with low sensitivity to CGRP8-37, this antagonist displaced [125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4 degrees C to 25 degrees C, suggesting the anomalous behaviour of CGRP8-37 is not due to temperature differences between binding and functional assays.

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 microM) 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 microM) 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 microM 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 microM AM22-52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73+/-0.14). BIBN4096BS (10 microM) 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 approximately betaCGRP>alphaCGRP. 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 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.

Neuropeptides in drug research.

Neuropeptides have been a subject of considerable interest in the pharmaceutical industry over the last 20 years or more. Many drug discovery teams have contributed to our understanding of neuropeptide biology but no significant drugs that act selectively upon neuropeptide receptors have yet emerged from the clinic. There are, however, a plethora of clinically useful drugs that act at other classes of neurotransmitter and neuromodulator receptors, many of them discovered over the last 20 years. Nevertheless, we think that the future for the discovery of novel drugs acting at neuropeptide receptors looks bright for two reasons: (1) there has been a substantial increase in our understanding of the function of neuropeptides; and (2) high-throughput screening (HTS) against neuropeptide receptors has now begun to yield many interesting drug-like molecules, rather than peptides, that have the potential to become clinically useful drugs. The objective of this review is to summarise our current understanding of specific areas of neuropeptide biology and pharmacology in the CNS as well as the PNS. We will also speculate on where we think the new generation of neuropeptide agonists and antagonists could emerge from the clinic.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates.

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast.

BACKGROUND AND PURPOSE: The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. EXPERIMENTAL APPROACH: We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. KEY RESULTS: The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. CONCLUSIONS AND IMPLICATIONS: These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future.

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. EXPERIMENTAL APPROACH: Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. KEY RESULTS: An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. CONCLUSIONS AND IMPLICATIONS: RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function.

BACKGROUND AND PURPOSE: Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC(2)) and the type 1 corticotrophin releasing factor receptor (CRF(1)) has been examined. EXPERIMENTAL APPROACH: GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by ELISA. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca(2+) mobilization and GTPγS binding to G(s), G(i), G(12) and G(q) were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2(+/-) mice was assessed. KEY RESULTS: The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC(2) enhanced the cell surface expression of all three RAMPs. CRF(1) enhanced the cell surface expression of RAMP2; the cell surface expression of CRF(1) was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF(1) : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2(+/-) mice, there was a loss of responsiveness to CRF. CONCLUSIONS AND IMPLICATIONS: The VPAC(2) and CRF(1) receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF(1), coupling to RAMP2 may be of physiological significance.

Lifting the lid on GPCRs: the role of extracellular loops.

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G-protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.