Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-(,) to G(i)-, and to G(q)-coupled effector signaling
Oa. Lawler et al., Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-(,) to G(i)-, and to G(q)-coupled effector signaling, J BIOL CHEM, 276(36), 2001, pp. 33596-33607
The prostacyclin receptor (IP) is primarily coupled to G alpha (s)-dependen
t activation of adenylyl cyclase; however, a number of studies indicate tha
t the IP may couple to other secondary effector systems perhaps in a specie
s-specific manner. In the current study, we investigated the specificity of
G protein:effector coupling by the mouse (m) IP overexpressed in human emb
ryonic kidney 293 cells, and endogenously expressed in murine, erythroleuke
mia cells. The mIP exhibited efficient G alpha (s) coupling and concentrati
on-dependent increases in cAMP generation in response to the IP agonist cic
a-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 stat
us. In addition, the mIP coupled to phospholipase C (PLC) activation in a p
ertussis toxin-insensitive, G alpha (i)-, G beta gamma-, and PKC-independen
t but in a G alpha (q)- and PKA-dependent manner. Whole cell phosphorylatio
n assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphor
ylation. 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 ph
osphorylation confirming that Ser(357) is the target residue for PKA-depend
ent phosphorylation. Finally, co-precipitation experiments permitted. the d
etection of G alpha (S), G alpha (i), and G alpha (q) in the immunoprecipit
ates of mIP, whereas only G alpha (i) was co-precipitated with mIP(S357A) i
ndicating 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 m
IP, in addition to coupling to G alpha (s), couples to G alpha (i) and G al
pha (q); however, G alpha (i) and G alpha (q) coupling is dependent on init
ial cicaprost-induced mIP:G alpha (s) coupling and phosphorylation of mIP b
y cAMP-dependent PKA where Ser(357) was identified as the target residue fo
r PKA phosphorylation.