Serine 331 is the major site of receptor phosphorylation induced by agentsthat activate protein kinase G in HEK 293 cells overexpressing thromboxanereceptor alpha
S. Yamamoto et al., Serine 331 is the major site of receptor phosphorylation induced by agentsthat activate protein kinase G in HEK 293 cells overexpressing thromboxanereceptor alpha, ARCH BIOCH, 393(1), 2001, pp. 97-105
Human embryonic kidney (HEK)293 cells stably transfected with the His-tagge
d thromboxane receptor alpha (TP alpha) was used to study the phosphorylati
on and desensitization of the receptor induced by 8-bromocyclic GMP (8-Br-c
GMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These age
nts are known to activate cGMP-dependent protein kinase (PKG). Pretreatment
of cells with these agents attenuated significantly agonist I-BOP induced
Ca2+ release. These agents also induced dose-dependent phosphorylation of t
he TPa as demonstrated by increased P-32-labeling of the receptor from cell
s prelabeled with (32)Pi. To facilitate the identification of the intracell
ular domains involved in phosphorylation, glutathione S-transferase (GST)-i
ntracellular domain fusion proteins were used as substrates for the purifie
d PKG. It was found that only the GST-C-terminal tail fusion protein could
serve as a substrate for the PKG. To identify the specific serine/threonine
residues in the C-terminal tail being phosphorylated, various alanine muta
nts of these serine/threonine residues were checked for their ability to se
rve as substrates. It was found that the Ser-331 of the C-terminal tail was
primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a
predominant site of phosphorylation was supported by in vivo studies in whi
ch HEK293 cells expressing the S331A mutant receptor showed little phosphor
ylation induced by any of the above three agents. Furthermore, HEK293 cells
expressing the S331A mutant receptor pretreated with any of the above thre
e agents became responsive to the agonist I-BOP-induced Ca2+ release. These
results indicate that Ser-331 of the TPa is the primary site responsible f
or the phosphorylation and the desensitization of the receptor induced by a
gents that activate the PKG. (C) 2001 Academic Press.