Abstract

The prostaglandin E-prostanoid (EP)₃ receptor signals primarily through the inhibitory G protein G_i, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP₃ receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP₃ receptor splice variants to modulate expression of a β-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in β-galactosidase enzymatic activity with EC₅₀ ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp³³⁸ with Ala, or Arg³²⁹ with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in β-galactosidase activity, whereas substitution of Lys³⁰⁰ with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonG_i/G_o pathway is activated by the EP₃ receptor. Direct measurement 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 EP₃ receptor signals through a novel cAMP response element binding protein/CRE pathway.