Protein Kinase A and assembly of an ABCC4 protein network.

Cyclic AMP (cAMP) compartmentalization at the plasma membrane employs export by ABCC4, yet the protein machinery that creates spatially restricted signaling has remained unclear. We show that robust PKA activation increases ABCC4 at the cell surface and assembles a PDZ-dependent macromolecular complex that constrains ABCC4 mobility and stabilizes the transporter. Using Avi-tag crosslinking/AP-MS and APEX proximity labeling, we map a PKA-induced ABCC4 neighborhood enriched for PDZ-domain scaffolds that couple ABCC4 to actin and cell junctions. SCRIB emerges as a previously unrecognized interactor whose depletion lowers surface ABCC4 and blunts ABCC4-dependent drug resistance. Deleting the ABCC4 PDZ motif accelerates lateral diffusion ~4-fold, halves protein half-life, and attenuates PKA-stimulated increases in surface ABCC4 and cAMP efflux, establishing a requirement for the PDZ motif in optimal export under PKA drive. Finally, the selective ABCC4 inhibitor Ceefourin-2 lowers ABCC4's melting temperature and dismantles the actin/junction-enriched neighborhood, revealing a non-classical small molecule inhibitory mechanism based on network disruption. These findings define a PKA-driven, PDZ-dependent ABCC4 protein neighborhood that stabilizes the transporter and optimizes cAMP efflux-an advance over prior work that localized ABCC4 but lacked a mechanistic blueprint for its membrane microdomain organization.