Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Abstract

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP 3R) is an intracellular Ca 2+ channel which plays a major role in Ca 2+ signalling. Three isoforms of IP 3R have been identified (IP 3R-1, IP 3R-2 and IP 3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP 3R are poorly known. RINm5F cells who express almost exclusively (∼90%) the IP 3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP 3R-3-mediated Ca 2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP 3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP 3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP 3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca 2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca 2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca 2+ signalling cascade, whereby activated PKC dampens IP 3R-3 activity.