Abstract
We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLC-PK(1). Determination of the amino acid sequence and cDNA analysis encoding a CRSP precursor showed that this peptide has approximately 60% identity in the amino acid sequence with human calcitonin gene-related peptide type-alpha (alphaCGRP), type-beta (betaCGRP), and porcine CGRP. Northern blot analysis and radioimmunoassay demonstrated that CRSP is expressed mainly in the thyroid gland and the central nervous system, in which the calcitonin receptor was abundantly expressed. Synthetic CRSP elicited a potent stimulatory effect on the cAMP production in LLC-PK(1) cells. Although it shows significant sequence similarity with CGRPs, this peptide did not elicit cAMP elevation in cells that endogenously expressed a CGRP receptor or an adrenomedullin receptor or were transfected with either of these recombinant receptors. Administration of CRSP into anesthetized rats did not alter the blood pressure but induced a transient decrease in the plasma calcium concentration. In fact, this peptide potently increased the intracellular cAMP concentration in COS-7 cells that expressed the recombinant calcitonin receptor. These unique properties indicate that CRSP is not a porcine counterpart of betaCGRP and probably elicits its biological effects via the calcitonin receptor.
Highlights
We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLCPK1
We designated this peptide as calcitonin receptor-stimulating peptide (CRSP)
The peptide fraction obtained was separated by Sephadex G-25 gel filtration, and each fraction was assayed by monitoring the cAMP production in LLC-PK1 cells
Summary
Cell Culture—LLC-PK1, Hs68, Swiss 3T3, and COS-7 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum supplemented with 100 g/ml penicillin and 100 units/ml streptomycin in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. Fractions containing peptides of Mr 3000 were pooled and subjected to carboxymethyl (CM) ion exchange chromatography (CM-52, 24 ϫ 450 mm; Whatman) eluting with a linear gradient elution of HCOONH4 (pH 6.5) from 10 mM to 1 M in the presence of 10% CH3CN. The biologically active fractions were separated by reverse phase HPLC on a C18 column (218TP54, 4.6 ϫ 250 mm; Vydac), and the peptide was purified on a diphenyl column (219TP5215, 2.1 ϫ 150 mm; Vydac) using a linear gradient elution of CH3CN from 10 to 60% in 0.1% trifluoroacetic acid. The homogenate was centrifuged, and the resulting supernatant was lyophilized and dissolved in the same volume of RIA buffer (50 mM sodium phosphate (pH 7.4) containing 80 mM NaCl, 25 mM EDTA, 0.05% NaN3, 0.5% BSA treated with N-ethylmaleimide, and 0.5% Triton X-100). Spectra were measured in a multi-channel acquisition mode in the mass range m/z 300 –2000 with scan durations of 3.5 s
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