Roman Muff

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.

An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3.

Human receptor activity modifying proteins (RAMP) regulate the ligand specificity of the calcitonin-receptor-like-receptor (McLatchie et al., Nature 393:333-339 (1998)). Here we have investigated binding of [125I]-labeled human (h) calcitonin ([125I]hCT) and rat amylin ([125I]amylin) to rabbit aortic endothelial cells (RAEC) co-transfected with the hCT receptor isotype 2 (hCTR2) and RAMP1, -2 or -3. Specific binding of 125 pM [125I]hCT to cells transfected with hCTR2 alone was 6.7 +/- 0.7 fmol/50,000 cells (n=5), and was reduced by 45 +/- 2% and 86 +/- 3% (P < 0.001) in the presence of RAMP1 and -3, but remained unchanged with RAMP2. In the absence and presence of individual RAMPs [125I]hCT binding inhibition occured with similar IC50 of between 6 nM and 11 nM hCT, and human amylin was 24- to 54-fold less potent. Specific binding of 125 pM [125I]amylin to cells transfected with hCTR2 alone was 0.9 +/- 0.2 fmol/50,000 cells (n=6), and was increased by 262 +/- 48% (P < 0.005), 73 +/- 26% (P < 0.05) and 338 +/- 57% (P < 0.005) with RAMP1, -2 or -3, respectively. [125I]amylin binding was inhibited with IC50 of 3.1 +/- 0.5 nM and 4.0 +/- 0.8 nM human amylin in cells co-transfected with RAMP1 or -3, respectively, and hCT was 45 +/- 2- and 126 +/- 3-fold less potent. In conclusion, RAMP1 and -3 decrease calcitonin receptor expression in RAEC transfected with hCTR2 encoding cDNA and simultanously reveal an amylin receptor.

A Receptor Activity Modifying Protein (RAMP)2-Dependent Adrenomedullin Receptor Is a Calcitonin Gene-Related Peptide Receptor when Coexpressed with Human RAMP11

Adrenomedullin (ADM) and α- and β-calcitonin (CT) gene-related peptide (α-, βCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and[ 125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable[ 125I]hαCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in[ 125I]rADM binding inhibition experim...

Calcitonin gene-related peptide is a stimulator of renin secretion.

Calcitonin gene-related peptide (CGRP) was found to stimulate renin secretion in vivo in normal human volunteers. Moreover, CGRP stimulated the release of renin in vitro from isolated rat renal juxtaglomerular cells (half-maximal effective concentration [EC50] 100 nM) concomitant with stimulation of cAMP production (EC50 60 nM). Immunoreactive CGRP was recognized in rat renal cortical nerve fibers, and intact rat CGRP was identified in extracts of the rat renal cortex. Because CGRP containing sensory nerve fibers are seen in the region of the juxtaglomerular apparatus, it would seem that the release of CGRP from these afferent nerves may be involved in the physiological control of renin secretion.

Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma × glioma hybrid cells (NG108-15)

Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [125I]ADM(1-52) as radioligand. Half-maximal inhibition of [125I]ADM(1-52) binding by intact rat ADM(1-50) amounted to 0.27 +/- 0.03 nM (n = 15). The related peptides rat alpha-CGRP, rat amylin and salmon calcitonin displaced [125I]ADM(1-52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of cAMP accumulation by rat ADM(1-50) was obtained with 1.00 +/- 0.12 nM (n = 16). Rat alpha-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma x rat glioma hybrid cells (NG108-15), but here rat alpha-CGRP was > 1000-fold less potent than rat ADM(1-50). Human ADM(22-52) and human CGRP-I(8-37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1-50) stimulated cAMP accumulation with inhibitory constants of 365 +/- 93 nM and 92 +/- 2 nM In astrocytes, and 45 +/- 3 nM and 1300 +/- 500 nM in NG108-15 cells. Rat ADM(1-50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as truncated receptor antagonists ADM(22-52) and CGRP(8-37) in rat astrocytes and neuroblastoma x glioma hybrid cells are consistent with ADM receptor isotypes in the brain.

Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells

Inhibition of human [125I]calcitonin gene-related peptide-I ([125I]hCGRP-I) binding by human adrenomedullin (hADM), its N-terminal truncated fragments, CGRP and amylin, and cyclic AMP accumulation were examined in the human neuroblastoma cell line SK-N-MC. Binding of [125I]hCGRP-I (125 pM) was inhibited by hCGRP-I, hADM(1-52), hADM(13-52), and human amylin with IC50 of 0.32 +/- 0.06, 2.11 +/- 0.26, 3.45 +/- 0.54, and 68.8 +/- 6.6 nM, respectively. hCGRP-I(8-37) and hADM(22-52), which lack the N-terminal ring structure, inhibited [125I]hCGRP-I binding with IC50 of 2.35 +/- 0.45 and > 1000 nM. hCGRP-I, hADM(1-52), hADM(13-52) and human amylin stimulated cAMP accumulation with EC50 of 0.40 +/- 0.05, 18.1 +/- 2.6, 51.3 +/- 9.0 and 925 +/- 159 nM, respectively. hCGRP-I(8-37) (100 nM) antagonized hCGRP-I and hADM(1-52) stimulated cAMP production with the same Ki of 16.6 +/- 1.2 and 16.8 +/- 1.1 nM. In conclusion, human ADM, which is more distantly related to CGRP than amylin, interacts more potently with the CGRP receptor in SK-N-MC cells than amylin. The N-terminal ring structure of hADM, unlike that of hCGRP, is essential for binding to the CGRP receptor. Coupling of hADM binding to cAMP stimulation is less efficient than for hCGRP-I and is reduced by deletion of the unique 12 amino acid sequence of hADM N-terminal to the ring structure.

Receptor activity modifying proteins regulate the activity of a calcitonin gene-related peptide receptor in rabbit aortic endothelial cells

In Xenopus oocytes with an endogenous calcitonin gene‐related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333–339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) αCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist αCGRP(8–37). Co‐expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to hαCGRP by 47% (P<0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22–52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.

Regulation of hormone secretion and cytosolic Ca2+ by extracellular Ca2+ in parathyroid cells and C-cells: Role of voltage-sensitive Ca2+ channels

The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.

Biological importance of the peptides of the calcitonin family as revealed by disruption and transfer of corresponding genes

The hormone calcitonin (CT) of thyroid C-cell origin, the neuropeptides alpha- and beta-calcitonin gene-related peptide (CGRP), the widely expressed hormone and tissue factor adrenomedullin (AM), and amylin (AMY) that is co-produced with insulin in pancreatic beta-cells, are structurally related peptides. They have in common six or seven amino acid ring structures, linked by disulfide bridges between cysteine residues, and amidated carboxyl termini that are both required for biological activity. The actions of the peptides in vivo have traditionally been studied after intravenous and intracerebroventricular administration. As a result, CT lowers serum calcium and reduces pain perception. alpha- and beta CGRP and AM are highly potent vasodilatory peptides. AMY inhibits food intake through its action in the area postrema of the brain. Physiological actions of the peptides summarized in the present review have been defined through gene knockout and overexpression strategies.

Cathepsins and osteosarcoma: Expression analysis identifies cathepsin K as an indicator of metastasis

Osteosarcoma is the most frequent malignant bone tumor with a poor survival rate for patients with metastasis. Previous studies have shown that beside other proteases, distinct sets of cathepsins are involved in the process of metastasis of different tumors. In this study we investigated the expression of cathepsin proteases in human osteosarcoma metastasis. First, the mRNA expression of 14 human cathepsins was studied in SAOS‐2 osteosarcoma cells and the highly metastatic LM5 and LM7 sublines by reverse transcriptase (RT)‐polymerase chain reaction (PCR). The expression of cathepsin D, K, and L mRNA was found upregulated and that of cathepsin F, H, and V downregulated in the highly metastatic LM5 and LM7 cells. A subgroup of the cathepsin proteases was further studied at the protein level by Western blot analysis of cell extracts. The expression of cathepsin B and H was decreased and that of cathepsin D, K, and L was increased in the highly metastatic cell lines as compared to the SAOS‐2 cell line. Diagnostic relevance of cathepsin K expression in osteosarcoma was revealed upon correlation of survival and metastasis with immunohistochemical cathepsin K staining of biopsies collected from 92 patients prior to chemotherapy. Patients with metastatic high‐grade osteosarcoma and low cathepsin K expression at diagnosis had a better prognosis than those with high expression. Thus, it appears that cathepsin K expression is of predictive prognostic value for patients with high‐grade tumors and metastasis at diagnosis.