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Structure and methylation of the human calcitonin/alpha-CGRP gene.

ABSTRACT: We report a detailed analysis of the human calcitonin/alpha-CGRP gene locus. About 39kb of DNA containing the gene has been mapped and a common Pvu II RFLP identified downstream of the gene. DNA sequence analysis revealed an extensive CpG island containing several rare restriction enzyme sites at the 5' end of the gene. The structure of this island is unusual in that it contains two distinct CpG-rich regions, one located around exon 1 and the other about 1.5kb further upstream. Msp I sites within both CpG-rich regions were found to be unmethylated, regardless of whether the calcitonin/alpha-CGRP gene was being expressed. However, a correlation was found between demethylation of Msp I sites in intron 2, downstream of the CpG island, and calcitonin/alpha-CGRP gene expression. DNA sequence analysis also revealed the presence of several binding sites for constitutive and regulatory transcription factors in the promoter of the gene. These results suggest that both unmethylated CpG islands and specific demethylation of internal sequences may play a role in the activation of calcitonin/alpha-CGRP gene transcription.

SUBMITTER: Broad PM

PROVIDER: S-EPMC318429 | biostudies-other | 1989 Sep

REPOSITORIES: biostudies-other

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Structure and methylation of the human calcitonin/alpha-CGRP gene.

Broad P M PM Symes A J AJ Thakker R V RV Craig R K RK

Nucleic acids research 19890901 17

We report a detailed analysis of the human calcitonin/alpha-CGRP gene locus. About 39kb of DNA containing the gene has been mapped and a common Pvu II RFLP identified downstream of the gene. DNA sequence analysis revealed an extensive CpG island containing several rare restriction enzyme sites at the 5' end of the gene. The structure of this island is unusual in that it contains two distinct CpG-rich regions, one located around exon 1 and the other about 1.5kb further upstream. Msp I sites withi ...[more]

PMID: 2571128

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Project description:Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

Project description:: Human α-calcitonin gene-related peptide (h-α-CGRP) is a highly potent vasodilator peptide that belongs to the family of calcitonin peptides. There are two forms of CGRP receptors in humans and rodents: α-CGRP receptor predominately found in the cardiovascular system and β-CGRP receptor predominating in the gastrointestinal tract. The CGRP receptors are primarily localized to C and Aδ sensory fibers, where they are involved in nociceptive transmission and migraine pathophysiology. These fibers are found both peripherally and centrally, with extensive perivascular location. The CGRP receptors belong to the class B G-protein-coupled receptors, and they are primarily associated to signaling via Gα proteins. The objectives of the present work were: (i) synthesis of three single-labelled fluorescent analogues of h-α-CGRP by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis, and (ii) testing of their biological activity in isolated human, mouse, and rat arteries by using a small-vessel myograph setup. The three analogues were labelled with 5(6)-carboxyfluorescein via the spacer 6-aminohexanoic acid at the chain of Lys24 or Lys35. Circular dichroism (CD) experiments were performed to obtain information on the secondary structure of these fluorescently labelled peptides. The CD spectra indicated that the folding of all three analogues was similar to that of native α-CGRP. The three fluorescent analogues of α-CGRP were successfully prepared with a purity of >95%. In comparison to α-CGRP, the three analogues exhibited similar efficacy, but different potency in producing a vasodilator effect. The analogue labelled at the N-terminus proved to be the most readily synthesized, but it was found to possess the lowest vasodilator potency. The analogues labelled at Lys35 or Lys24 exhibited an acceptable reduction in potency (i.e., 3-5 times and 5-10 times less potent, respectively), and thus they have potential for use in further investigations of receptor internalization and neuronal reuptake.

Dataset Information

Structure and methylation of the human calcitonin/alpha-CGRP gene.

Publications

Structure and methylation of the human calcitonin/alpha-CGRP gene.

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