Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3, respectively) from mouse lung and found a novel alternative splicing segment, SI _m2, at 176-208 of IP₃R2. The long form (IP₃R2 SI_m2⁺) was dominant, but the short form (IP₃R2 SI_m2^-) was detected in all tissues examined. IP ₃R2 SI_m2^- has neither IP₃ binding activity nor Ca²⁺ releasing activity. In addition to its reticular distribution, IP₃R2 SI_m2⁺ is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca²⁺ ionophore stimulation, most of the IP₃R2 SI_m2⁺ is in clusters. IP₃R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca²⁺ ionophore stimulation. IP₃R2 SI _m2^- does not form clusters in either resting or stimulated cells. IP₃ binding-deficient site-directed mutants of IP ₃R2 SI_m2⁺ and IP₃R3 fail to form clusters, indicating that IP₃ binding is involved in the cluster formation by these isoforms. Coespression of IP₃R2 SI _m2^- prevents stimulus-induced IP₃R clustering, suggesting that IP₃R2 SI_m2^- functions as a negative coordinator of stimulusinduced IP₃R clustering. Expression of IP₃R2 SI_m2^- in CHO-K1 cells significantly reduced ATP-induced Ca²⁺ entry, but not Ca²⁺ release, suggesting that the novel splice variant of IP₃R2 specifically influences the dynamics of the sustained phase of Ca²⁺ signals.