Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila.
AU - Isaac, R. Elwyn
AU - Johnson, Erik C.
AU - Audsley, Neil
AU - Shirras, Alan D.
PY - 2007/12/15
Y1 - 2007/12/15
N2 - Recent studies have firmly established pigment dispersing factor (PDF), a C-terminally amidated octodecapeptide, as a key neurotransmitter regulating rhythmic circadian locomotory behaviours in adult Drosophila melanogaster. The mechanisms by which PDF functions as a circadian peptide transmitter are not fully understood, however; in particular, nothing is known about the role of extracellular peptidases in terminating PDF signalling at synapses. In this study we show that PDF is susceptible to hydrolysis by neprilysin, an endopeptidase that is enriched in synaptic membranes of mammals and insects. Neprilysin cleaves PDF at the internal Ser7–Leu8 peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila PDF receptor cDNA and a firefly luciferase reporter gene, confirming that such cleavage results in PDF inactivation. The Ser7–Leu8 peptide bond was also the principal cleavage site when PDF was incubated with membranes prepared from heads of adult Drosophila. This endopeptidase activity was inhibited by the neprilysin inhibitors phosphoramidon (IC50, 0.15 µmol l–1) and thiorphan (IC50, 1.2 µmol l–1). We propose that cleavage by a member of the Drosophila neprilysin family of endopeptidases is the most likely mechanism for inactivating synaptic PDF and that neprilysin might have an important role in regulating PDF signals within circadian neural circuits.
AB - Recent studies have firmly established pigment dispersing factor (PDF), a C-terminally amidated octodecapeptide, as a key neurotransmitter regulating rhythmic circadian locomotory behaviours in adult Drosophila melanogaster. The mechanisms by which PDF functions as a circadian peptide transmitter are not fully understood, however; in particular, nothing is known about the role of extracellular peptidases in terminating PDF signalling at synapses. In this study we show that PDF is susceptible to hydrolysis by neprilysin, an endopeptidase that is enriched in synaptic membranes of mammals and insects. Neprilysin cleaves PDF at the internal Ser7–Leu8 peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila PDF receptor cDNA and a firefly luciferase reporter gene, confirming that such cleavage results in PDF inactivation. The Ser7–Leu8 peptide bond was also the principal cleavage site when PDF was incubated with membranes prepared from heads of adult Drosophila. This endopeptidase activity was inhibited by the neprilysin inhibitors phosphoramidon (IC50, 0.15 µmol l–1) and thiorphan (IC50, 1.2 µmol l–1). We propose that cleavage by a member of the Drosophila neprilysin family of endopeptidases is the most likely mechanism for inactivating synaptic PDF and that neprilysin might have an important role in regulating PDF signals within circadian neural circuits.
KW - pigment dispersing factor
KW - neprilysin
KW - peptidase
KW - neuropeptide
KW - circadian rhythm
U2 - 10.1242/jeb.012088
DO - 10.1242/jeb.012088
M3 - Journal article
VL - 210
SP - 4465
EP - 4470
JO - Journal of Experimental Biology
JF - Journal of Experimental Biology
SN - 0022-0949
IS - 24
ER -