Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca2+ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase-anchoring proteins). In many cell types, the activation of InsP(3)R (inositol 1,4,5-trisphosphate receptor), an endoplasmic reticulum Ca2+ channel, is a key event of Ca2+ Signalling. The phosphorylation of InsP(3)R1 by PKA stimulates Ca2+ mobilization. This control is thought to be tight, involving the association of PKA with InsP(3)R1. The InsP(3)R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP(3)R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP(3)R1 associated with PKA type II beta and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co-purification of AKAP450 with InsP(3)R1 on heparin agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP(3)R1 and RII beta (regulatory subunit of PKA II beta) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP(3)R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP(3)R1 forms a complex with AKAP450 and PKAII beta, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP(3)R1 with PKA in Purkinje cells would require a different macromolecular complex.