ABCC4 protein interactions using IP-MS/MS

The ATP-binding cassette transporter ABCC4 regulates cAMP membrane microdomain formation after adenylate cyclase activation but how ABCC4 localizes to these membrane microdomains are still unclear. Herein we demonstrate that the assembly and spatiotemporal organization of ABCC4’s protein neighborhood by PKA requires its PDZ motif and PKA activation. Density gradient ultracentrifugation revealed the formation of ABCC4 PDZ motif–specific, large protein complex upon PKA activation. To elucidate how PKA ushers ABCC4 to form this membrane supercomplex we identified proteins that closely interact with ABCC4 and proteins in ABCC4’s “neighborhood” network using Avi-tagged or APEX-tagged ABCC4 constructs, and their deletion mutants without the PDZ motif. We found that ABCC4 interacts with numerous PDZ domain containing proteins (PDZ proteins), and the PKA-induced enrichment of actin filament-binding proteins in the vicinity of ABCC4 requires the PDZ motif. The assembly of the supercomplex was crucially dependent on the actin-binding protein Moesin (MSN), which also interacts with the PDZ motif. This supercomplex can be disrupted by a specific potent inhibitor of ABCC4. These findings indicate that PKA activation promotes ABCC4 membrane localization by enhancing the assembly of a protein scaffold through PDZ proteins, MSN and actin cytoskeletal proteins.