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The stimulation of mitogenic signaling pathways by N-POMC peptides.

Pepper DJ, Bicknell AB - Mol. Cell. Endocrinol. (2008)

Bottom Line: However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC(1-28) stimulates the ERK pathway in human H295R cells.Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC(1-49) stimulated the phosphorylation of Akt more robustly than N-POMC(1-28).Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK.

ABSTRACT
The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC(1-28) stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC(1-28) and N-POMC(1-49) in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC(1-49) stimulated the phosphorylation of Akt more robustly than N-POMC(1-28). We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal.

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PCR using degenerate oligonucleotides of genes containing the tyrosine kinase catalytic domain in Y1 cells and also in normal rat adrenal gland. The expected band size of approx 200 bp was excised, cloned and 114 clones of each sequenced.
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fig4: PCR using degenerate oligonucleotides of genes containing the tyrosine kinase catalytic domain in Y1 cells and also in normal rat adrenal gland. The expected band size of approx 200 bp was excised, cloned and 114 clones of each sequenced.

Mentions: The identity of the receptor for N-POMC remains elusive. In an attempt to isolate possible candidate molecule we used PCR with degenerate oligonucleotide primers to amplify the conserved sequence within the catalytic domains of receptor-type tyrosine kinase molecule as has been described previously (Tanaka et al., 1998). Using this approach with cDNA derived from both Y1 cells and also from rat adrenal tissue we successfully amplified a band of the expected 200 bp (Fig. 4). The bands were excised and cloned into pGEM-T and colonies picked for plasmid isolation and subsequent sequencing. One hundred and fourteen clones from each cDNA were successfully sequenced. Every clone represented a tyrosine kinase molecule, although many of them were found not to be receptors (Table 1).


The stimulation of mitogenic signaling pathways by N-POMC peptides.

Pepper DJ, Bicknell AB - Mol. Cell. Endocrinol. (2008)

PCR using degenerate oligonucleotides of genes containing the tyrosine kinase catalytic domain in Y1 cells and also in normal rat adrenal gland. The expected band size of approx 200 bp was excised, cloned and 114 clones of each sequenced.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651482&req=5

fig4: PCR using degenerate oligonucleotides of genes containing the tyrosine kinase catalytic domain in Y1 cells and also in normal rat adrenal gland. The expected band size of approx 200 bp was excised, cloned and 114 clones of each sequenced.
Mentions: The identity of the receptor for N-POMC remains elusive. In an attempt to isolate possible candidate molecule we used PCR with degenerate oligonucleotide primers to amplify the conserved sequence within the catalytic domains of receptor-type tyrosine kinase molecule as has been described previously (Tanaka et al., 1998). Using this approach with cDNA derived from both Y1 cells and also from rat adrenal tissue we successfully amplified a band of the expected 200 bp (Fig. 4). The bands were excised and cloned into pGEM-T and colonies picked for plasmid isolation and subsequent sequencing. One hundred and fourteen clones from each cDNA were successfully sequenced. Every clone represented a tyrosine kinase molecule, although many of them were found not to be receptors (Table 1).

Bottom Line: However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC(1-28) stimulates the ERK pathway in human H295R cells.Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC(1-49) stimulated the phosphorylation of Akt more robustly than N-POMC(1-28).Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK.

ABSTRACT
The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC(1-28) stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC(1-28) and N-POMC(1-49) in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC(1-49) stimulated the phosphorylation of Akt more robustly than N-POMC(1-28). We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal.

Show MeSH
Related in: MedlinePlus