cAMP-dependent PKA Signaling Induces TNFR1 Exosome-like Vesicle Release via Anchoring of PKA Regulatory Subunit RIIβ to BIG2

The 55-kDa, type I tumor necrosis factor receptor (TNFR1) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide-exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosomelike vesicles via an ARF1and ARF3dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-Br-cAMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a PKAdependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIβ modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIβ to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIβ to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotideexchange protein that activates class I ARFs and as an AKAP for RIIβ that localizes PKA signaling within cellular TNFR1 trafficking pathways.