Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins

Abstract

IRBIT [inositol 1,4,5-trisphosphate receptor (IP 3 R) binding protein released with inositol 1,4,5-trisphosphate (IP 3 )] is a multifunctional protein that regulates several target molecules such as ion channels, transporters, polyadenylation complex, and kinases. Through its interaction with multiple targets, IRBIT contributes to calcium signaling, electrolyte transport, mRNA processing, cell cycle, and neuronal function. However, the regulatory mechanism of IRBIT binding to particular targets is poorly understood. Long-IRBIT is an IRBIT homolog with high homology to IRBIT, except for a unique N-terminal appendage. Long-IRBIT splice variants have different N-terminal sequences and a common C-terminal region, which is involved in multimerization of IRBIT and Long-IRBIT. In this study, we characterized IRBIT and Long-IRBIT splice variants (IRBIT family). We determined that the IRBIT family exhibits different mRNA expression patterns in various tissues. The IRBIT family formed homo-and heteromultimers. In addition, N-terminal splicing of Long-IRBIT changed the protein stability and selectivity to target molecules. These results suggest that N-terminal diversity of the IRBIT family and various combinations of multimer formation contribute to the functional diversity of the IRBIT family.IRBIT | Long-IRBIT | splice variant | protein stability | protein-protein interaction

Both Irbit and Long-Irbit Bind to and Coordinately Regulate Cl-/hco3-Exchanger Ae2 Activity Through Modulating the Lysosomal Degradation of Ae2.

“…However, given that authentic L-IRBIT (L-IRBIT V1 or L-IRBIT V2) was predominantly expressed in B16-F10 cells, and that L-IRBIT KO cells were produced using a guide RNA sequence that targets a common exon of all L-IRBIT variants, our results likely reflect the effects of IRBIT and authentic L-IRIBT. However, as shown in a previous report 12 , short variants of L-IRBIT show a more marked cell type-specific expression. Thus, especially in some cell types, the contribution of short-form L-IRBIT variants to AE2 regulation should be verified in future studies.…”

“…At the cellular level, however, L-IRBIT shows a more restricted expression pattern than IRBIT, for example, the specific expression in some interneurons of the cerebellum 9 . A recent report of splicing variants of L-IRBIT with different N-terminal sequences has shown that each variant has unique properties in protein stability, target molecule spectrum, and functional consequence 12 . Together, IRBIT and L-IRBIT can form homo-or hetero-multimers via conserved AHCY domains.…”

“…Together, IRBIT and L-IRBIT can form homo-or hetero-multimers via conserved AHCY domains. The "IRBIT family" is supposed to serve as a hub protein and to be involved in diverse cellular functions, especially in the regulation of the intracellular ionic milieu 12 .…”

Aberrant IP 3 receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29

“…We constructed an expression vector encoding human IP 3 R1 (SI − /SII + /SIII − splicing variant) (33) with an N-terminal mRFP-P2A tag. The peptide bond at the C terminus of the P2A peptide is cleaved in cells, and mRFP fluorescence can be used as a marker of transfected cells (36). We generated 14 missense mutants of human IP 3 R1: R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, V479I, E497K, T579I, N587D, S1478D, and V1538M, identified in SCA29, SCA15, and ataxic cerebral palsy (2-10, 13, 32) ( Fig.…”

Consensus report of the 8 and 9th Weinman Symposia on Gene x Environment Interaction in carcinogenesis: novel opportunities for precision medicine

“…IRBIT was discovered as a molecule that binds to the IP 3 -binding pocket of IP 3 R [146,147]. In contrast, an IRBIT homologue Long-IRBIT does not interacts with IP 3 R1 because its long N-terminal appendage inhibits the interaction [148,149]. IRBIT is a multifunctional protein that regulates diverse proteins including Na + -HCO 3 co-transporter, CFTR, Na + -H + exchanger [150], CaM kinase IIα [151], and PIP kinases [152].…”