Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to Gs-, to Gi-, and to Gq-coupled effector signaling

The prostacyclin receptor (IP) is primarily coupled to G alpha (s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells, and endogenously expressed in murine, erythroleukemia cells. The mIP exhibited efficient G alpha (s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cica-prost; however, mIP also coupled to G alpha (i) decreasing the levels of cAMP in forskolin-treated. cells. mIP coupling to G alpha (i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha (i)-, G beta gamma-, and PKC-independent but in a G alpha (q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha (s) but failed to couple to G alpha (i) or to efficiently couple to G alpha (q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted. the detection of G alpha (S), G alpha (i), and G alpha (q) in the immunoprecipitates of mIP, whereas only G alpha (i) was co-precipitated with mIP(S357A) indicating that Ser (357) of mIP is essential for G alpha (i) and G alpha (q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha (i) or G alpha (q) Taken together our data indicate that the mIP, in addition to coupling to G alpha (s), couples to G alpha (i) and G alpha (q); however, G alpha (i) and G alpha (q) coupling is dependent on initial cicaprost-induced mIP:G alpha (s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.