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

Abstract

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