Abstract
Inositol 1,4,5-trisphosphate receptors (InsP(3)R) play a key role in intracellular calcium (Ca(2+)) signaling. Three InsP(3)R isoforms are expressed in mammals. Type 1 InsP(3)R (InsP(3)R1) is a predominant neuronal isoform. Neuronal InsP(3)R1 is one of the major substrates of protein kinase A (PKA) phosphorylation. In our previous study (Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J. Neurosci. 23, 403-415) we discovered a direct association between InsP(3)R1 and protein phosphatase 1 alpha (PP1 alpha). In functional experiments we demonstrated that phosphorylation by PKA activates InsP(3)R1 and that dephosphorylation by PP1 alpha inhibits InsP(3)R1. To extend these findings, here we investigated the possibility of InsP(3)R1-PKA association. In a series of biochemical experiments we demonstrate the following findings. 1) InsP(3)R1 and PKA associate in the brain. 2) InsP(3)R1-PKA association is mediated by the AKAP9 (Yotiao) multi-functional PKA anchoring protein. 3) InsP(3)R1-AKAP9 association is mediated via the leucine/isoleucine zipper (LIZ) motif in the InsP(3)R1 coupling domain and the fourth LIZ motif in AKAP9. 4) The InsP(3)R association with AKAP9 is specific for type 1 InsP(3)R. 5) Both the SII(+) and the SII(-) coupling domain splice variants of InsP(3)R1 bind to AKAP9. 6) Binding to AKAP9 promotes association of neuronal InsP(3)R1 with the NR1 NMDA receptor; and 7) neuronal InsP(3)R1 associate with PP1 directly via carboxy-terminus and indirectly via AKAP9. The obtained results advance our understanding of cross-talk between cAMP and InsP(3)/Ca(2+) signaling pathways in the brain.
Highlights
In the previous paper we demonstrated the direct association of neuronal InsP3R type 1 (InsP3R1) with the catalytic subunit of protein phosphatase 1␣ (PP1␣) [16]
We discovered a novel InsP3R1-AKAP9-protein kinase A (PKA) complex in the brain and demonstrated that InsP3R1-AKAP9 association mediated by leucine/isoleucine zipper (LIZ) motifs in InsP3R1 and AKAP9 (Yotiao)
InsP3R1-PKA Association Involves an Adaptor Protein and LIZ Motif in the InsP3R1 Sequence—To determine whether InsP3R1 and PKA form a complex in the brain, we performed a series of InsP3R1 immunoprecipitation experiments with rat brain lysates
Summary
Plasmid Construction—GST fusion proteins in the pGEX-KG expression vector ϭ GST-RT1-LIZ ϭ 1251–1287 of rat InsP3R1 [18], and GST-RT3-LIZ ϭ 1242–1278 of rat InsP3R3 [19]. Synaptosomes (P2) were prepared from rat brains as described previously [24], solubilized in extraction buffer A, clarified by centrifugation, (100,000 ϫ g in TL-100), and immunoprecipitated with an anti-InsP3R1 polyclonal antibody attached to protein A-Sepharose beads. Precipitation of [3H]InsP3 Binding Sites—Rat brain lysates, prepared as described above, were immunoprecipitated with anti-InsP3R1, antiAKAP9, or anti-AKAP6 rabbit polyclonal antibodies attached to protein A-Sepharose beads. 72 h after transfection, cells were collected with ice-cold PBS and solubilized for 30 min at 4 °C in extraction buffer containing 0.5% Triton X-100, 20 mM imidazole, pH 6.8, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors. Extracts were clarified by centrifugation for 20 min at 100,000 ϫ g (TL-100 rotor, Beckman), mixed with Sf9 cells lysates containing recombinant InsP3R, incubated for 2 h at 4 °C, and precipitated by isoform-specific anti-InsP3R polyclonal antibodies. Rabbit polyclonal anti-AKAP9 (anti-Yotiao) antibody (ZMD.252) is from Zymed Laboratories Inc
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.