Tm. Palmer et Gl. Stiles, Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: Evidence for regulation by-multiple protein kinase C isoforms, BIOCHEM, 38(45), 1999, pp. 14833-14842
Activation of the AZA adenosine receptor (A(2A)AR)contributes to the neurom
odulatory and neuroprotective effects of adenosine in the central nervous s
ystem. Here we demonstrate that, in rat C6 glioma cells stably expressing a
n epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and thr
eonine residues can be increased by pretreatment with either the synthetic
protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or e
ndothelin I, which increases PKC activity via binding to endogenous endothe
lin(A) receptors. Under conditions in which PMA was maximally effective, ac
tivation of other second messenger-regulated kinases was without effect. Wh
ile basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR
-selective: antagonist ZM241385, they were both blocked by GF109203X (a sel
ective inhibitor of conventional and novel-PKC isoforms) and rottlerin (a P
KC delta-selective inhibitor) but not Go6976 (selective for conventional PK
C isoforms). However, coexpression of the A(2A)AR with each of the a, PI, a
nd PII isoforms of PKA increased basal and PMA-stimulated phosphorylation.
Mutation of the three consensus PKC phosphorylation sites within the recept
or (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PM
A-stimulated phosphorylation. In addition, phosphorylation of the receptor
was not associated with detectable-changes in either its signaling capacity
or cell surface expression. These observations suggest that multiple PKC i
soforms can stimulate A(2A)AR phosphorylation via activation of one or more
downstream kinases which then phosphorylate the receptor directly. In addi
tion, it is likely that phosphorylation controls interactions with regulato
ry proteins distinct from those involved in the classical cAMP signaling pa
thway utilized by this receptor.