Lawler, Orlaith A., Miggin, Sinead and Kinsella, B. Therese (2001) 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*. Journal of Biological Chemistry, 276 (36). pp. 33596-33607. ISSN 0021-9258
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Abstract
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.
Item Type: | Article |
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Keywords: | Protein; Kinase; A-mediated Phosphorylation; serine 357; mouse; Prostacyclin Receptor; coupling; Gs-; Gi-; Gq-; Effector Signalling; |
Academic Unit: | Faculty of Science and Engineering > Biology |
Item ID: | 12645 |
Identification Number: | 10.1074/jbc.M104434200 |
Depositing User: | Sinead Miggin |
Date Deposited: | 27 Mar 2020 11:43 |
Journal or Publication Title: | Journal of Biological Chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology |
Refereed: | Yes |
Related URLs: | |
URI: | https://mural.maynoothuniversity.ie/id/eprint/12645 |
Use Licence: | This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here |
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