FIGURE 7.
Constitutive and cAMP-mediated TNFR1 exosome-like vesicle release requires the anchoring of RIIβ to BIG2 AKAP domains B and C. HUVEC were transfected with plasmids encoding either His-tagged wild-type BIG2 (His-BIG2) or BIG2 mutants that contained single amino acid substitutions in AKAP domain B (His-BIG2(V289W)), domain C (His-BIG2(V534W)), or domains B and C (His-BIG2(V289W/V534W)) or empty plasmid (Control), for 2 days before the addition of fresh, exosome-depleted medium for 24 h, without or with 1 mm 8-Br-cAMP (cAMP). A, proteins immunoprecipitated (IP) with the anti-His antibody (Pull-down) or remaining in the supernatant were immunoblotted with antibodies against RIIβ or the His tag. This blot is representative of three individual experiments. B, TNFR1 concentration in medium was quantified by ELISA. *, significant increase (His-BIG2, p < 10-3) or decrease (His-BIG2(V289W), p < 10-3; His-BIG2(V534W), p < 0.002; His-BIG2(V289W/V534W), p < 10-3) in the quantity of TNFR1 constitutively released into the medium as compared with control cells transfected with the empty plasmid (n = 6). **, significant increase (His-BIG2, p < 0.012) or decrease (His-BIG2(V289W), p < 10-4; His-BIG2(V534W), p < 10-4; His-BIG2(V289W/V534W), p < 10-4) in the quantity of TNFR1 released into the medium following stimulation with 1 mm 8-Br-cAMP for 24 h as compared with control cells transfected with the empty plasmid (n = 6). The quantity of constitutive and 8-Br-cAMP-mediated TNFR1 release from cells transfected with the domain B/C double mutant (His-BIG2(V289W/V534W)) was significantly decreased as compared with those transfected with His-BIG2(V289W) (p < 10-3) or His-BIG2(V534W) (p < 10-3), respectively (n = 6). C, representative Western blot, from one of three experiments, showing TNFR1 in medium and cell lysates, and BIG2 and β-tubulin in cell lysates. Images were grouped from adjacent parts of the same gel in A and C.