Mitochondrial bound PKA contributes to cyclic-AMP compartmentation in human airway smooth muscle cells.

Abstract

In human airway smooth muscle (hASM) cells, compartmentation of cAMP signaling is necessary to maintain the fidelity of responses by different G protein-coupled receptors sharing this common diffusible second messenger. Using FRET-based biosensors targeted to different subcellular locations, we found that the EP2 prostaglandin receptors (EP2Rs) stimulate cAMP production that is restricted to locations associated with non-lipid raft regions of the plasma membrane. Furthermore, inhibition of phosphodiesterase type 4 (PDE4) allowed EP2R production of cAMP to spread to subcellular locations associated with lipid-raft membrane domains. However, computational modeling has suggested that PDEs may only contribute to compartmentation if cAMP diffusion is slowed by some other means. To test this hypothesis, we used raster image correlation spectroscopy (RICS) to estimate the diffusion coefficient of fluorescently labeled cAMP (φ575-cAMP) in hASM cells and found that it is 3.9 ± 0.49 μm2/s, or nearly two orders of magnitude slower than the predicted rate of free diffusion. Disrupting protein kinase A (PKA) interactions with A kinase anchoring proteins (AKAPs) using the anchoring inhibitor Ht31 nearly doubled the diffusion coefficient of φ575-cAMP to 7.9 ± 1.5 μm2/s, suggesting that cAMP movement was being buffered by PKA anchored by one or more AKAPs. Confocal imaging demonstrated that φ575-cAMP colocalizes with mitochondria, and Western blot analysis found that D-AKAP2 is the primary mitochondrial AKAP found in hASM cells. Knockdown of D‑AKAP2 expression was achieved using adenovirus-mediated delivery of shRNA, and in knockdown cells EP2R activation was able to stimulate cAMP production in subcellular locations associated with lipid-raft domains, similar to effects previously seen with PDE4 inhibition. These results suggest that buffering of cAMP movement by PKA anchored to the outer membrane of mitochondria contributes to compartmentation of EP2R-mediated responses in hASM cells.