Biosynthesis of opioid peptides.
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Amino Acid Sequence↗
Biomedical subjects
E Herbert
Publications and source records attributed to E Herbert.
Use of gene transfer techniques to study neuropeptide gene regulation.
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Animals↗
Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing.
A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.
Adrenocorticotropic Hormone↗
Nucleotide sequence of cloned cDNA for pro-opiomelanocortin in the amphibian Xenopus laevis.
The function of alpha-melanocyte-stimulating hormone (alpha-MSH) is not known in mammals. It is well-established in the amphibian Xenopus laevis in which alpha-MSH mediates the process of adaptation to a dark background. The amino acid sequence of this hormone is, however, not known in amphibians. In order to determine the primary structure of the precursor protein for alpha-MSH, which in mammals has been called pro-opiomelancortin (POMC), we constructed a cDNA library from Xenopus pituitary mRNA. A pool of synthetic oligodeoxyribonucleotide tetradecamers corresponding to part of the mammalian alpha-MSH sequence was used to screen the library. The nucleotide sequence of a 1050-base pair hybridization-positive cDNA clone was determined and the deduced amino acid sequence of Xenopus POMC revealed the sequences of Xenopus gamma-MSH, alpha-MSH, corticotropin-like intermediate lobe peptide, beta-MSH, and beta-endorphin. Interestingly, the N-terminal amino acid of Xenopus alpha-MSH, which is N alpha-acetylated in the biologically active form of the hormone, is different from that of mammalian alpha-MSH. The distribution of the bioactive domains within Xenopus POMC is remarkably similar to that in other known POMC molecules and as in mammals the domains in the amphibian prohormone are flanked on both sides by pairs of basic amino acids.
Amino Acid Sequence↗
Sequence and expression of the rat prodynorphin gene.
We report here the isolation of a lambda genomic clone that contains the nucleotide sequence coding for the main exon of the rat prodynorphin (proenkephalin B) gene. This exon codes for the majority of the translated region of prodynorphin mRNA including the opioid peptides alpha-neo-endorphin, dynorphin A, and dynorphin B. The entire 3' untranslated region is also contained on the lambda clone. Nucleotide sequence comparison with the main exon of the human prodynorphin gene reveals both structural and sequence homology. RNA blot analysis reveals that prodynorphin transcripts can be seen in numerous regions of the rat brain and in the adrenal gland, spinal cord, testis, and anterior pituitary.
Animals↗
Isolation and characterization of the rat proenkephalin gene.
The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.
Amino Acid Sequence↗
5' sequence of porcine and rat pro-opiomelanocortin mRNA. One porcine and two rat forms.
We have determined the nucleotide sequences of the 5' noncoding regions and the regions encoding the first 56 amino acids of rat and porcine pro-opiomelanocortin (POMC) mRNA. We accomplished this by sequencing cDNA produced by elongating specific DNA primers hybridized to neurointermediate pituitary mRNA. The nucleotide sequence of the 5' region of rat POMC mRNA fills a gap in our knowledge of the structure of this mRNA and the protein it codes for. While we have observed only a single porcine POMC mRNA, two different rat POMC mRNAs are detected. The rat POMC mRNAs differ by a 30-base insertion/deletion in their 5' noncoding regions. Its position and sequence suggest that it is the result of alternate modes of intron removal during RNA splicing.
Animals↗
Regulation of expression of opioid peptide genes.
In the past three years it has been shown by recombinant DNA approaches that there are at least three different genes that code for opioid peptides. The basic structures of the three polyprotein precursor molecules from which bioactive opioid peptides are derived are remarkably similar. There are also similarities in the structure of the genes that code for these precursor molecules. Using immunological techniques, it has been shown that the levels of the opioid peptides can be regulated by altering the rates of protein processing or secretion. Recently, complementary DNA clones of the opioid peptide precursor molecules have been used as hybridization probes to determine that regulation also occurs at the level of gene expression (transcription of the opioid peptide genes).
Adrenalectomy↗
Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.
Exposure of bovine chromaffin cells in primary culture to 5 microM reserpine or 25 microM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 microM forskolin (or cholera toxin at 50 micrograms/ml) causes a 1.5- to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 microgram/ml). Reserpine (5 microM) elicits a 1.5- to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Met]enkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Met]enkephalin pentapeptide and a concomitant decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAenk) by RNA blot hybridization with a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (less than 17 hr) 3- to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr period. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.
Adrenal Medulla↗
Polyprotein gene expression: generation of diversity of neuroendocrine peptides.
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Animals↗
The mouse genome contains two nonallelic pro-opiomelanocortin genes.
In the anterior pituitary pro-opiomelanocortin (POMC) is the protein precursor to both adrenocorticotropin and beta-lipotropin but in the intermediate pituitary POMC serves as the precursor to alpha-melanocyte-stimulating hormone and beta-endorphin. In addition, POMC expression in the anterior pituitary is inhibited by glucocorticoids but stimulated by corticotropin-releasing factor while POMC expression in the intermediate lobe is not responsive to glucocorticoids but is inhibited by dopamine. In this study we have asked whether tissue-specific processing and regulation of POMC could be related to the presence of more than one POMC gene. We report here that the mouse genome contains two POMC related gene sequences, alpha- and beta-POMC, that alpha-POMC is located on mouse chromosome 12 while beta-POMC is on a different chromosome, probably chromosome 19. Sequencing of phage lambda recombinants containing alpha- and beta-POMC sequences indicated that the alpha-POMC gene in mouse is very similar to the single POMC gene found in human, bovine, and rat genomes. The sequence of the mouse beta-POMC gene is quite different from that of the alpha-POMC gene. One important difference is that the beta-POMC gene has a translation stop signal in place of the first amino acid in beta-endorphin (Tyr). The beta-POMC gene has many features in common with the pseudogene of the beta-globin family.
Alleles↗
Complete amino acid sequence of mouse pro-opiomelanocortin derived from the nucleotide sequence of pro-opiomelanocortin cDNA.
Polyadenylated RNA was isolated from a mouse pituitary tumor cell line (AtT-20/D16v) which synthesizes and secretes adrenocorticotropic hormone and beta-endorphin. The RNA was used to construct a cDNA library by a double linker technique and the library was screened for pro-opiomelanocortin (POMC) sequences. One recombinant plasmid, pMKSU16, contained a 923-base pair insert comprising the entire POMC-coding sequence as well as 98 bases of 5' noncoding and 5 bases of 3' noncoding sequence. The protein sequence predicted by the cDNA shows mouse POMC to consist of 235 amino acids with seven potential tryptic cleavage sites consisting of pairs of basic amino acid residues. Comparison with the previously published bovine POMC sequence suggests certain regions of POMC are highly conserved between the two species, particularly the regions corresponding to the alpha-, beta-, and gamma-melanocyte-stimulating hormones.
Adrenocorticotropic Hormone↗
Glucocorticoids regulate proopiomelanocortin gene expression in vivo at the levels of transcription and secretion.
After adrenalectomy, the plasma levels of adrenocorticotropic hormone (corticotropin, ACTH)/endorphin peptides in rats rise dramatically in the first 4 hr while pituitary peptide levels fall sharply. Eight hours after adrenalectomy, plasma levels are near control values again but they then increase continuously over the next 8 days. Proopiomelanocortin (POMC) mRNA levels in the anterior pituitary (quantitated by hybridization with cloned POMC cDNA) increase 2-fold in the first 24 hours, reaching 15- to 20-fold the control level 18 days after adrenalectomy. When dexamethasone is administered to rats 8 days after adrenalectomy, the above events are reversed. Plasma ACTH falls to control levels within 2 hr whereas anterior pituitary POMC mRNA requires 5 days of treatment for return to control levels. The levels of POMC mRNA in the neurointermediate lobe and the hypothalamus are not altered by either treatment. Adrenalectomy increases transcription of the POMC gene in the anterior pituitary approximately 20-fold and halves transcription of the growth hormone gene within 1 hr of operation. Administration of dexamethasone immediately after adrenalectomy suppresses the increase in transcription of the POMC gene and increases the transcription of the growth hormone gene. Transcription of the POMC gene(s) in the neurointermediate lobe is not altered by either of these treatments.
Adrenalectomy↗
Primary structure of the human proenkephalin gene.
The nucleotide sequence of a 6.8-kb region of human DNA containing the proenkephalin gene and flanking regions is reported. The gene consists of four exons separated by three introns and spans approximately 5.3 kb of DNA. Location and identification of several repetitive DNA sequences within and flanking the gene are also described. The distribution of CpG dinucleotides as well as the extent of CpG methylation at several restriction sites within and surrounding the gene is also presented. The structural organization of the human proenkephalin gene exhibits striking similarities to the organization of the human pro-opiomelanocortin (POMC) gene. Nucleotide sequence homologies suggest that two opioid peptide precursors, proenkephalin and prodynorphin, may have arisen by duplication from a common ancestral gene.
5-Methylcytosine↗
Generation of diversity and evolution of opioid peptides.
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Adrenal Medulla↗
Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells.
Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.
Adrenocorticotropic Hormone↗
Detection and quantitation of pro-opiomelanocortin mRNA in pituitary and brain tissues from different species.
Pro-opiomelanocortin-like mRNA has been detected in anterior and neurointermediate lobes of the pituitary, hypothalamus, amygdala, and cerebral cortex of rats by Northern blot analysis using a 144-base-pair DNA hybridization probe complementary to the beta-lipotropin domain of pro-opiomelanocortin mRNA. The hybridizing species of RNA in the pituitary lobes and hypothalmus is approximately 1100 bases long whereas that in the amygdala and cerebral cortex is 1000 bases long. Solution hybridization analysis with this same probe indicates that pro-opiomelanocortin mRNA is 20 times as concentrated in the neurointermediate lobe as in the anterior lobe of the pituitary. In turn, the pro-opiomelanocortin mRNA levels in the anterior pituitary are 1 to 2 orders of magnitude higher than those in the brain tissues. These results support the hypothesis that adrenocorticotropic hormone/endorphin peptides previously detected in the hypothalamus and amygdala with immunological techniques are synthesized in these tissues from a precursor similar to pro-opiomelanocortin in the pituitary.
Animals↗
Primary structure of the human Met- and Leu-enkephalin precursor and its mRNA.
The nucleotide sequence of a complete cDNA copy of enkephalin precursor mRNA from human phaeochromocytoma is reported. The corresponding amino acid sequence shows that the precursor is 267 amino acids long and contains six interspersed Met-enkephalin sequences and one Leu-enkephalin sequence. Five of the seven enkephalins are flanked on both sides by pairs of basic amino acid residues. The precursors does not contain the sequences of the opioid peptides, dynorphin, alpha-neo-endorphin or beta-endorphin.
Amino Acid Sequence↗