Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, G alpha(s), we demonstrate that attenuation of AC activity by loss of G alpha(s) more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that G alpha(s) may also specifically associate with each AC.IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, G alpha s also associates with the AC.IP3R signaling junction.