Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM(2) receptor, whereas when paired with the calcitonin receptor AMY(3) receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT((a))). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM(2) and AMY(3(a)) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

Role of β3-adrenergic receptors in the action of a tumour lipid mobilizing factor

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10-7-10-5M) of the specific β3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOKI cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein-1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor. © 2002 The Cancer Research Campaign.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non-neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [ 125I]-AM binding sites (K(d) 79±9 pM and B(max) 571±34 fmol mg -1 protein) were more abundant than specific [ 125I]-CGRP binding sites (K(d) 12±0.7 pM and B(max) 32±2 fmol mg -1 protein) in embryonic spinal cord cells. 4 Specific [ 125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM(22-52)>rCGRPα>CGRP(8-37) ≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS). 5 Specific [ 125I]-CGRP binding was competed by rCGRPα>rAM≥ CGRP(8-37)≥BIBN4096BS>hAM(22-52). 6 Cellular levels of cAMP were increased by AM (pEC"5"0 10.2±0.2) and less potently by rCGRPα (pEC"5"0 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP(8-37) (pA"2 7.63±0.44) and hAM(22-52) (pA"2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP(8-37) (pA"2 7.41±0.15) and AM(22-52) (pA"2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA"2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP"1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM"1 (CLR/RAMP2) receptor subtype. © 2006 Nature Publishing Group All rights reserved.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

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.

Desensitisation of adrenomedullin and CGRP receptors

Adrenomedullin (AM), a potent vasoactive peptide, is elevated in certain disease states such as sepsis. Its role as a physiologically relevant peptide has been confirmed with the advent of the homozygous lethal AM peptide knockout mouse. So far, there have been few and conflicting studies which examine the regulatory role of AM at the receptor level. In this article, we discuss the few studies that have been presented on the desensitisation of AM receptors and also present novel data on the desensitisation of endogenous AM receptors in Rat-2 fibroblasts. © 2003 Elsevier Science B.V. All rights reserved.

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

1. The role of individual residues in the 8-18 helix of CGRP 8-37 in promoting high-affinity binding to CGRP 1 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. 3. Arg 11 and Arg 18 were replaced by serines to give [Ser 11.18]CGRP 8-37. These bound with pKi values <6 to SK-N-MC cells and had apparent pA 2 values of 5.81 ± 0.04 and 5.31 ± 0.11 on SK-N-MC and L6 cells. CGRP 8-37 had a pKi of 8.22 on SK-N-MC cells and pK b values on the above cell lines of 8.95±0.04 and 8.76±0.04. 3. The arginines were replaced with glutamic acid residues. [Glu 11]CGRP 8-37 had a pK b of 7.14±0.14 on SK-N-MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu 18]CGRP 8-37 had a pK b of 7.10±0.0.08 on SK-N-MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. 4. Leu 12, Leu 15 and Leu 16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA 2 values of [bpa 12]-, [bpa 15]- and [bpa 16]CGRP 8-37 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). 5. It is concluded that the Arg 11 and Arg 18 are involved in specific electrostatic interactions with other residues, either on the CGRP 1 receptors or elsewhere on CGRP 8-37. Leu 16 is in a conformationally restricted site when CGRP 8-37 binds to CGRP 1 receptors, unlike Leu 12 and Leu 15.

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.

International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors

The calcitonin family of peptides comprises calcitonin, amylin two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

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 (pD 2s for CGRP, amylin and adrenomedullin of 7.90 ± 0.11, 7.70 ± 0.19 and 7.25 ± 0.10 respectively). 3. CGRP 8-37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin 22-52 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 pIC 50 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 (pIC 50 = 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 CGRP 2 receptor on the vas deferens with low sensitivity to CGRP 8-37, this antagonist displaced [ 125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP 8-37 is not due to temperature differences between binding and functional assays.

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

1. 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). 2. 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. 3. 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. 4. 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. 5. On SK-N-MC cells, hαCGRP(8-18, 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. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.