Adenosine A2A receptors are predominantly expressed in the dendrites of enkephalin-positive γ-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A2A receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A2A receptor signaling in the striatum. Specifically, the adenosine A2A receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A2A receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-d-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, Mr 32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca2+/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A2A receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA substrates were unresponsive to this agent, consistent with the postsynaptic localization of adenosine A2A receptors. Finally, the phosphorylation state of these proteins was further assessed in vivo by systemic administration of caffeine. © 2006 Elsevier B.V. All rights reserved.