Elsevier

Gene

Volume 207, Issue 2, 30 January 1998, Pages 219-225
Gene

Short Communication
The isolation and characterization of the promoter of the human type 1 inositol 1,4,5-trisphosphate receptor

https://doi.org/10.1016/S0378-1119(97)00630-6Get rights and content

Abstract

In humans, at least three types of inositol (1,4,5)-trisphosphate receptor (IP3R) are present. The gene encoding type 1 IP3R (IP3R-I) is expressed in all cell types, although expression predominates in Purkinje cells. To study the regulation of the human IP3R-I gene, we isolated and characterized a 2.1-kb 5′ flanking region. In transient expression assays using a rat cell line, analysis of various deletion mutants demonstrated that a fragment of only 86 bp 5′ of the putative tsp displayed a promoter activity similar to that of the 2.1-kb fragment. Also, we compared the sequence of the human IP3R-I promoter with the sequence of the mouse IP3R-I promoter. Considerable sequence homology is present in four distinct domains, which include several conserved putative binding sites for transcription factors. Further, we demonstrate a decrease in the activity of the isolated human IP3R-I promoter and of the endogenous IP3R-I promoter after 48 h of treatment with retinoic acid. Analysis of deletion constructs of the human promoter indicates that the decreased promoter activity in response to retinoic acid is likely to be mediated by a conserved AP-2 binding site.

Introduction

Inositol (1,4,5)-trisphosphate (InsP3) is a second messenger formed in response to stimulation of a wide variety of seven transmembrane spanning receptors. InsP3 can bind to, and activate, a family of intracellular Ca2+ permeable channels, the InsP3 receptors (IP3Rs) (Berridge, 1993). Cloning and sequencing studies have shown that several types of IP3R exist. At least three types (IP3R-I, -II and -III) have been found in human (Maeda et al., 1989; Miyawaki et al., 1990; Yamada et al., 1994; Yamamoto-Hino et al., 1994), rat (Mignery et al., 1990) and mouse tissue (Marks et al., 1990; De Smedt et al., 1997).

Recent studies have shown that the ratios at which type I, II and III IP3Rs are expressed differ considerably between cell types (Sugiyama et al., 1994, De Smedt et al., 1994; Wojcikiewicz et al., 1995), on both protein and mRNA levels. IP3R-I is expressed in all cell types studied (De Smedt et al., 1994; Wojcikiewicz et al., 1995) and found at exceptionally high levels in Purkinje cells (Maeda et al., 1989). The expression of IP3R-II and -III isoforms has been found along with the IP3R-I isoform in many non-neural cell types (De Smedt et al., 1994; Wojcikiewicz et al., 1995). Besides differences between different cell types, a considerable degree of variance in relative mRNA levels of the three IP3R types has been observed in human myometrial (Morgan et al., 1996) and human atrial muscle (Deelman, unpublished). The widespread expression of IP3Rs underscores the important role of these receptors in cellular signalling. Nevertheless, little is known about significant functional differences of the isoforms, although it has been suggested that substrate affinity and possibly the interaction with cytoskeletal elements vary between the IP3R type expressed (White et al., 1993). At present, the molecular mechanisms responsible for gene expression of the different IP3R types are still unclear.

The aim of the present study was to isolate and characterize the human IP3R-I promoter and to compare its structure with the mouse IP3R-I promoter (Furutama et al., 1996).

Section snippets

Isolation and sequence of the 5′-flanking region of the human IP3R-I gene

The 5′-flanking region of the human IP3R-I gene was isolated using a PCR-based method (Fig. 1). The sequence data of the 2.1-kb fragment obtained from the EcoRV library were aligned with sequence data of the mouse IP3R-I promoter region (Furutama et al., 1996). The human IP3R-I promoter regions from −1497 to −1433, from −816 to −569, from −469 to −315 and from −149 to +145 are highly homologous to regions of the mouse IP3R-I promoter (Fig. 2). The TATA box and the CAAT box are well conserved

Conclusions

  • 1.

    We have cloned the promoter region of the human IP3R-I gene. Functional analysis in A7r5 cells has demonstrated that basal promoter activity of the human IP3R-I gene was contained in the region from −86 to +145 relative to the putative tsp.

  • 2.

    A comparison of the sequences of the human and mouse IP3R-I promoter revealed that the promoters share a high degree of homology in four domains. In particular, the area around the putative tsp is well conserved between species.

  • 3.

    The activity of the isolated

Unlinked references

Gidoni et al. (1984), Spandidos et al. (1989)listed but not cited—please check.

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