Visit our Expo - Redox and Inflammation signaling 2012

Session II : Receptor signaling and G proteins Poster II, 26
Molecular mechanism of CRF induced calcium mobilization in CRF1 and CRF2
expressing cells: involvement of different signalling pathways
Eric Gutknecht1-2, Ilse Van der Linden1, Georges Vauquelin2 and Frank M.
Dautzenberg1
1CNS Discovery, Johnson & Johnson Research & Development, Turnhoutsweg 30, 2340
Beerse and 2MBFA, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium
Corticotropin releasing factor (CRF), the most important mediator of the body’s stress
response activates two receptors (CRF1 and CRF2) both belonging to the B-class family of
the GPCRs. CRF1 and CRF2 mainly couple to the stimulatory G-protein Galpha s which
activates adenylate cyclase and protein kinase A. However, we reported recently that in stably
transfected HEK293 cells, both receptors induce transient calcium (Ca2+) mobilization in the
fluorimetric imaging plate reader (FLIPR) format. Ca2+ mobilization was completely
abolished after depletion of intracellular Ca2+ stores or inhibition of IP3 receptors. In
contrast, a specific inhibitor of the PKA pathway had no effect, whereas blockade of
phospholipase C abolished the Ca2+ mobilization. To better understand the involvement of
the various G-proteins, we incubated cells with pertussis toxin and cholera toxin, two well
known inhibitors of Gi and Gs proteins. While pertussis toxin partially blocked Ca2+ (40%),
cholera toxin almost completely (80%) inhibited the transient Ca2+ response. To further
assess the contribution of Galpha i in this process and the role of beta-gamma subunits, we
transiently transfected the C-terminal part of the G-protein receptor kinase type 2 GRK2 (as a
beta-gamma scavenger) which resulted in a potent reduction of the Ca2+ response. To
elucidate the mechanism of the phospholipase C activation, we transfected different alphasubunits
of the Gq family in CRF1 and CRF2(a) expressing cells. While a potentiation of the
Ca2+ signal was observed with Galpha-16, only a weak or no increase in the signal could be
monitored with Galpha o, Galpha olf, Galpha q, or Galpha 11. Thus, in CRF1 and CRF2
expressing HEK293 cells, both receptors seems to act through different G-proteins indicating
that several signalling pathways, including Galpha s, are involved in the activation of
phospholipase C.
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