We have previously shown that CFTR significantly interacts with adenosine receptors (A2BR) in the plasma membrane (1). This interaction leads to a subsequent CFTR-sensitive increase in adenosine-dependent cAMP signaling in non-cystic fibrosis cells and suggests that innate defense is impaired by the lack of CFTR due to reduced cAMP signaling. This enhancement appears to be specific for A2BR and in contrast, CFTR does not increase β2 adrenergic receptor (β2AR)-dependent cAMP production. Little is known regarding how CFTR and A2BR interact to form a macromolecular cluster. Since CFTR and A2BR are both indirectly connected to the actin cytoskeleton, we tested the hypothesis that CFTR’s enhancement of A2BR was dependent on C-terminal PSD-95, Dlg, ZO-1 (PDZ) domain interactions. All experiments were performed in HEK293T cells transiently transfected with cDNA constructs outlined below. FRET was measured using the acceptor-photobleaching method by confocal microscopy and cAMP was measured by ELISA as described (1). Values are means ± S.E.M. and compared by the Mann-Whitney U test. We used FRET to probe interactions between CFTRgfp and A2BRmCherry and measured 8.2±1.0% FRET in the plasma membrane with these constructs (p<0.05; n =32). CFTR’s C-terminus is involved in CFTR-protein interactions, so we disrupted CFTR’s C-terminal PDZ domain (L1480A) and looked at the effect on CFTR-A2BR FRET. The L1480A CFTR mutation significantly reduced CFTR/A2BR FRET to 2.8±0.4% (n=11), suggesting that PDZ binding is critical for CFTR-A2BR interactions. These decreases in FRET were mirrored by concomitant reductions in cellular cAMP production (all n=6 and p<0.05). We then generated new constructs where we switched the PDZ-binding domains of A2BR and β2AR and measured FRET and cAMP production. Placing the β2AR C-terminus on A2BR (A2BRDSLL) significantly reduced A2BR FRET (62% decrease; n=12) and also decreased cAMP production by a similar value. In contrast, placing A2BR’s C-terminus on β2AR (β2ARGVGL) significantly increased CFTR-β2AR FRET by 80% and also increased β2AR-induced cAMP production by 34% (all n=12). Importantly, these maneuvers reversed interactions and made A2BRDSLL cAMP production CFTR-insensitive and made β2ARGVGL cAMP production CFTR-sensitive. We conclude that PDZ-binding domains play a role in CFTR-A2BR interactions and in differentiating between β2AR and A2BR signaling to coordinate cAMP production.