Phosphorylation of type-1 inositol 1,4,5-trisphosphate receptors by cyclic nucleotide-dependent protein kinases: A mutational analysis of the functionally important sites in the S2⁺ and S2^- splice variants

Inositol 1,4,5-trisphosphate receptors (InsP₃R) are the major route of intracellular calcium release in eukaryotic cells and as such are pivotal for stimulation of Ca²⁺-dependent effectors important for numerous physiological processes. Modulation of this release has important consequences for defining the particular spatio-temporal characteristics of Ca²⁺ signals. In this study, regulation of Ca²⁺ release by phosphorylation of type-1 InsP₃R (InsP₃R-1) by cAMP (PKA)- and cGMP (PKG)-dependent protein kinases was investigated in the two major splice variants of InsP₃R-1. InsP₃R-1 was expressed in DT-40 cells devoid of endogenous InsP₃R. In cells expressing the neuronal, S2⁺ splice variant of the InsP₃R-1, Ca ²⁺ release was markedly enhanced when either PKA or PKG was activated. The sites of phosphorylation were investigated by mutation of serine residues present in two canonical phosphorylation sites present in the protein. Potentiated Ca²⁺ release was abolished when serine 1755 was mutated to alanine (S1755A) but was unaffected by a similar mutation of serine 1589 (S1589A). These data demonstrate that Ser-1755 is the functionally important residue for phosphoregulation by PKA and PKG in the neuronal variant of the InsP₃R-1. Activation of PKA also resulted in potentiated Ca ²⁺ release in cells expressing the non-neuronal, S2^- splice variant of the InsP₃R-1. However, the PKA-induced potentiation was still evident in S1589A or S1755A InsP₃R-1 mutants. The effect was abolished in the double (S1589A/S1755A) mutant, indicating both sites are phosphorylated and contribute to the functional effect. Activation of PKG had no effect on Ca²⁺ release in cells expressing the S2 ^--variant of InsP₃R-1. Collectively, these data indicate that phosphoregulation of InsP₃R-1 has dramatic effects on Ca ²⁺ release and defines the molecular sites phosphorylated in the major variants expressed in neuronal and peripheral tissues.