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 Signalling*

The prostacyclin receptor (IP) is primarily coupled to Gαs-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 Gαs coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G α (i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G α (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, α (i)-, G β γ -, and PKC-independent but in a Gαq- 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 Gαs but failed to couple to Gα(i) or to efficiently couple to Gα(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 Gαs, Gα(i), and Gα(q) in the immunoprecipitates of mIP, whereas only Gαs was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for Gα(i) and Gα(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with Gα(i) or Gα(q). Taken together our data indicate that the mIP, in addition to coupling to Gαs, couples to Gα(i) and Gα(q); however, Gα(i) and Gα(q) coupling is dependent on initial cicaprost-induced mIP:Gαs coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation

Investigation of a functional requirement for isoprenylation by the human prostacyclin receptor

In the current study, we have established that the human (h) prostacyclin receptor (IP) is isoprenylated in whole cells. Through site directed mutagenesis and generation of the isoprenylation defective hIPSSLC, it was established that while isoprenylation of hIP does not influence ligand binding, it is obligatory for agonist activation of adenylyl cyclase and cAMP generation. Overexpression of GαS significantly augmented cAMP generation by the hIP but not by the hIPSSLC. Moreover, GαS co-immunoprecipitated with hIP following agonist activation but did not co-immunoprecipitate with hIPSSLC. Whereas hIP mediated concentration-dependent activation of phospholipase C (PLC); the extent of PLC activation by hIPSSLC was impaired compared to hIP. Co-expression of Gαq significantly augmentated intracellular calcium mobilization by the hIP but not by hIPSSLC. Moreover, whereas Gαq co-immunoprecipitated with hIP, it failed to co-immunoprecipitate with hIPSSLC. While both the hIP and hIPSSLC underwent agonist-induced internalization, the kinetics and extent of hIPSSLC internalization was impaired compared to hIP. Altering the CAAX motif of the hIP from a farnesyl (–CSLC) to a geranylgeranyl (–CSLL) isoprene acceptor, to generate hIPCSLL, did not affect ligand binding and yielded a receptor that exhibited identical signalling through both Gs- and Gq-coupled effectors to that of hIP. Thus, whereas isoprenylation of hIP does not influence ligand binding, it is functionally imperative in regulating post-receptor events including agonist-activation of adenylyl cyclase, for efficient activation of PLC and for receptor internalization. Though the nature of the isoprenoid attached to hIP does not act as a major determinant, the presence of an isoprenoid group, for example farnesyl or geranylgeranyl, is required for functional receptor–G protein interaction and coupling and for efficient agonist- induced receptor internalization