Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kDa protein: characterization of the determinants of structural specificity

Abstract

We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-delta 1 [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P3. Deletion of N-terminal residues 116-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.

GABAAReceptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

“…PRIP-1 is a novel Ins(1,4,5)P 3 binding protein, which has a number of binding partners, including GABARAP and PP1␣ (Takeuchi et al, 1996(Takeuchi et al, , 1997Yoshimura et al, 2001;. Although the precise cellular function of PRIP-1 remains unknown, deletion of this protein in mice produces GABA A receptors with modified pharmacological properties and behavior, suggesting a role for PRIP-1 in facilitating receptor assembly or activity .…”

“…Phospholipase C (PLC)-related inactive protein type 1 (PRIP-1) is a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] binding protein (Kanematsu et al, 1992;Yoshida et al, 1994), which is homologous to PLC-␦1 but catalytically inactive (Kanematsu et al, 1992Yoshida et al, 1994;Takeuchi et al, 1996Takeuchi et al, , 1997Takeuchi et al, , 2000. PRIP-1 has a number of binding partners, including catalytic subunit of protein phosphatase 1␣ (PP1␣) and GABA A receptor-associated protein (GABARAP) Yoshimura et al, 2001; Kanematsu et al, 2002).…”

d - myo -Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways

“…Because our data indicate that the effect of Ins(1,4,5,6)P 4 on Cl Ϫ secretion was downstream of PtdIns3K, it is possible that Ins(1,4,5,6)P 4 competes with PtdInsP 3 for effector binding sites because Ins(1,4,5,6)P 4 is a partial structural analog of the PtdInsP 3 headgroup (27). As a result, Ins(1,4,5,6)P 4 could compete with phosphoinositide binding to pleckstrin homology domains of signaling proteins and inhibit the normal recruitment of signal transduction complexes to the plasma membrane (28,29).…”

Interaction of p130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1α

“…The identification of a p130-related molecule in such a simple organism as C. elegans suggests that this family of proteins diverged early from other PLC isozymes. We propose that this distinct family of PLC-related proteins be designated the PLC-related catalytically inactive protein (PRIP) family (comprising PRIP-1 and -2 subfamilies).To investigate the physiological functions of PRIP family proteins, we previously examined the possible role of the binding of inositol compounds to the PH domain of p130 (10,(15)(16)(17). Our results suggested that p130, which is localized predominantly in the cytoplasm, contributes to Ins(1,4,5)P 3 -mediated Ca 2ϩ signaling.…”