Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element-mediated gene transcription

The prostaglandin E-prostanoid (EP), receptor signals primarily through the inhibitory G protein G(i), thereby decreasing intracellular cAMP levels. T o study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A a nd the novel splice Variant NT, which lacks the C-terminal sequence. The ab ility of the EP3 receptor splice variants to modulate expression of a beta- galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprost one-mediated increase in beta-gatactosidase enzymatic activity with EC50 ra nging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice va riant. Substitution of either Asp(338) with Ala, or Arg(329) with Ala or Gl u in the 77A splice variant resulted in a loss of receptor-evoked increases in P-galactosidase activity, whereas substitution of Lys(300) with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transducti on was insensitive to pretreatment of cells with pertussis toxin, suggestin g that a nonG(i)/G(o) pathway is activated by the EP3 receptor. Direct meas urement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP, recept or signals through a novel cAMP response element binding protein/CRE pathwa y.