Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2006) 11 P389

ECE2006 Poster Presentations Diabetes, metabolism and cardiovascular (174 abstracts)

Multiple signalling pathways are involved in the phosphorylation of ERK1/2 upon activation of human OX1R and OX2R: Evidence for differential modulation by orexin-A and orexin-B

J Tang , J Chen , H Lehnert & HS Randeva


Biological Sciences, University of Warwick, Coventry, United Kingdom.


Orexin-A (OR-A) and orexin-B (OR-B) play an important role in the regulation of energy balance and the control of sleep-wake cycle. They act via G-protein coupled receptors, namely orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). OX2R has equal affinity for both OR-A and OR-B, whilst OX1R has a 10-fold greater affinity for OR-A. Orexin-mediated functions have been extensively explored, however, the intracellular signalling pathways remain poorly understood. Using HEK-293 cells, we investigated the signalling pathways involved in extracellular-regulated-kinase 1 and 2 (ERK1/2) were mediated by OX1R and OX2R activation.

Full-length human OX1R and OX2R were amplified from human brain by RT-PCR and subsequently cloned and transfected into HEK-293 cells. Using OR-A and OR-B (0.01, 0.1, 1, 10 and 100 nM), phosphorylation of ERK1/2 were examined time course (5, 10, 15, 20 and 30 min). These effects were further dissected by the use of specific inhibitors for PKA and PKC and Gi/o-protein blocker (pertussis toxin, PTX).

The results demonstrated a dose-dependent phosphorylation and activation of ERK1/2 with a maximal activation by OR-A and OR-B (5 and 15 min). Activation of ERK1/2 by OR-A was greater than OR-B in HEK293-OX1R transfected cells, whereas a similar level of ERK1/2 activation by OR-A or OR-B was observed in HEK293-OX2R transfected cells. In HEK293-OX1R and –OX2R transfected cells, OR-A- and OR-B-induced ERK1/2 activation was abolished by PKC inhibition. Inhibition of PKA also attenuated OR-A- and OR-B-induced ERK1/2 activation. However, the effect was weaker than that observed for PKC. This suggests that PKC is mainly involved in OX1R- and OX2R–mediated ERK1/2 activation. In addition, orexin-activated phosphorylation of ERK1/2 was inhibited with pre-treatment of PTX in HEK293-OX2R transfected cells, but not in HEK293-OX1R transfected cells.

In conclusion, activation of ERK1/2 by OR-A an OR-B via both receptors resulted in a dose–dependent response with a maximal activation between 5 and 15 min. These data demonstrated that PKC and PKA signalling pathways are involved in the phosphorylation of ERK1/2 via OX1R and OX2R. Furthermore OX2R activation to ERK1/2 is associated with PTX-sensitive G-protein.

Volume 11

8th European Congress of Endocrinology incorporating the British Endocrine Societies

European Society of Endocrinology 
British Endocrine Societies 

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