Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

Activation of A(3) adenosine receptors (A(3)ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A(3)AR agonist stimulation of SERT activity is lost in A(3)AR knockout mice. A(3)AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A(3)ARs target SERTs among other potential effectors is unknown. Here we present evidence that A(3)ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A(3)AR/hSERT cotransfected cells. Treatment of A(3)AR/SERT-cotransfected Chinese hamster ovary cells with the A(3)AR agonist N-6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 mu M, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A(3)AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A(3)AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A(3)AR activation. Because PKGI and nitric-oxide synthetase are required for A(3)AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A(3)AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.