Cellular expression and subcellular localization of the human Ins(1,3,4,5)P(4)-binding protein, p42(IP4), in human brain and in neuronal cells.
In this study we describe for the human inositol-(1,3,4,5)-tetrakisphosphate (InsP4)-binding protein, p42IP4, the cellular distribution and subcellular localization in human brain and in transfected neuronal cells. The cDNA sequence of the human p42IP4 containing a single open reading frame yields a peptide of 374 amino acids with a calculated molecular mass of 43.4 kDa with a zinc-finger motif at the N-terminus, followed by two pleckstrin homology (PH) domains. Using a peptide-specific antiserum, p42IP4 protein was localized in a majority of neuronal cells of human brain sections. In the hypothalamus a subpopulation of paraventricular and infundibular nucleus neurons were strongly immunoreactive for p42IP4. In cortical areas the protein was predominantly found in large pyramidal cells. Some immunoreactivity for p42IP4 was also observed in the pyramidal cells of the hippocampal formation. Functional expression of p42IP4 protein in neuronal (NG108-15) and non-neuronal (CHO-K1) cells stably transfected with GFP-p42IP4 was shown in all cell fractions (homogenate, cytosol and membranes) by specific [3H]Ins(1,3,4,5)P4 binding activity, which correlated with p42IP4 protein detection by Western blot analysis. Using confocal laser scanning microscopy we showed that in NG108-15 and CHO-K1 cells stably transfected with GFP-p42IP4 the full length p42IP4 protein was localized in the cytoplasm, at the plasma membrane and in the nucleus. A deletion mutant of p42IP4 lacking the zinc finger domain resulted in solely a cytosolic and membrane localization but was not found in the nucleus. Thus we can conclude that human p42IP4 shows a region-specific localization in the human brain and the zinc finger motif within the protein is responsible for the localization of the protein in the cell nucleus.