Two Functions for IP3 Receptor Partner

Science's STKE  27 Jun 2006:
Vol. 2006, Issue 341, pp. tw211
DOI: 10.1126/stke.3412006tw211

Inositol 1,4,5-trisphosphate (IP3), a second messenger formed in response to stimulation of cell surface receptors, binds to and activates IP3 receptors (IP3Rs, internal calcium release channels) to liberate calcium from intracellular stores. Phosphorylated IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) binds to the IP3-binding region of the IP3R, from which it is displaced by IP3. Ando et al., who initially identified IRBIT, used an in vitro binding assay to show that IRBIT displaced IP3 from a fusion protein containing the IP3R lacking its channel domain. Scatchard analysis indicated that IRBIT acted as a competitive inhibitor that decreased the affinity of the IP3R for IP3, and mutational analysis indicated that most of the amino acid residues necessary for IP3 binding to the IP3R were also involved in IRBIT binding. Both IRBIT that had been exposed to alkaline phosphatase and a mutant form (S68A, in which serine 68 was substituted with alanine) that showed decreased phosphorylation and less efficient binding to the IP3R than wild-type IRBIT failed to suppress IP3 binding to the IP3R. IRBIT also inhibited IP3 binding and IP3-dependent calcium release in cerebellar microsomes. Calcium imaging of HeLa cells in which IRBIT was depleted with siRNA revealed enhanced release of calcium compared with wild-type cells in response to stimulation with an IP3-generating agonist. Overexpression of S68A (which bound to endogenous IRBIT, forming a heteromultimer with weaker affinity for the IP3R than that of the wild-type homomultimer) also enhanced IP3-dependent release. Thus, the authors propose that IRBIT acts as a "pseudoligand" regulated by phosphorylation that inhibits IP3 binding and thereby calcium release.

In a second paper, another group from the Mikoshiba lab proposes a second function of IRBIT based on the idea that when IP3 binds to IP3Rs, displaced IRBIT might serve to carry a signal to other partners. Shirakabe et al. therefore searched for proteins that interacted with IRBIT in extracts of mouse cerebellum. A prominent interacting protein from membrane fractions was identified as NBC1, the Na+/HCO3 cotransporter that shuttles HCO3 and Na+ ions across the plasma membrane. Reciprocal immunoprecipitation experiments verified that the endogenous proteins interact. Like its binding to the IP3R, binding of IRBIT to NBC1 was dependent on phosphorylation of IRBIT. The functional consequence of the interaction was tested in a Xenopus oocyte system. Electrophysiological measurements showed that expression of IRBIT along with NBC1 was necessary to allow full activity of the transporter. IRBIT interacted only with the NBC1 splicing variant called pNBC1, which is present in the pancreas, where it is thought to promote HCO3 transport in pancreatic duct cells. Thus, the authors propose that, at least in the pancreas, physiological concentrations of IP3, which cause dissociation of IRBIT from IP3Rs, could not only promote release of calcium through the IP3R channel but also mobilize an IRBIT-mediated signal that modulates acid-base balance.

H. Ando, A. Mizutani, H. Kiefer, D. Tsuzurugi, T. Michikawa, K. Mikoshiba, IRBIT suppresses IP3 receptor activity by competing with IP3 for the common binding site on the IP3 receptor. Mol. Cell 22, 795-806 (2006). [Online Journal]

K. Shirakabe, G. Priori, H. Yamada, H. Ando, S. Horita, T. Fujita, I. Fujimoto, A. Mizutani, G. Seki, K. Mikoshiba, IRBIT, an inositol 1,4,5-trisphosphate receptor-binding protein, specifically binds to and activates pancreas-type Na+/HCO3 cotransporter 1 (pNBC1). Proc. Natl. Acad. Sci. U.S.A. 103, 9542-9547 (2006). [Abstract] [Full Text]