Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity

Spatiotemporal regulation of c AMP within the cell is required to achieve receptor‐specific responses. The mechanism through which the cell selects a specific response to newly synthesized c AMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent c AMP ‐responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) c AMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein‐coupled receptor (Ado‐ GPCR ), bound to either A 2A or A 2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in c AMP levels after receptor activation, and its participation was confirmed by antibody‐mediated inactivation. A 2A ‐Ado‐ GPCR selective stimulation activates adenylyl cyclase 6 ( AC 6), which is bound to AKAP 79/150, to synthesize c AMP which is used by two other AKAP 79/150‐tethered proteins: protein kinase A ( PKA ) and phosphodiesterase 3A ( PDE 3A). In contrast, A 2B ‐Ado‐ GPCR stimulation activates D‐ AKAP 2‐attached AC 5 to generate c AMP , which is channeled to two other D‐ AKAP 2‐tethered proteins: guanine‐nucleotide exchange factor 2 (Epac2) and PDE 3B. In both cases, prior activation of PKA or Epac2 with selective c AMP analogs prevents de novo c AMP synthesis. In addition, we show that c AMP does not diffuse between these protein macrocomplexes or ‘signalosomes’. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered c AMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.