Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

A.H Sharp,S.H Snyder,S.K Nigam

doi:10.1016/s0021-9258(18)42537-9

A.H Sharp, S.H Snyder + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)42537-9

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Abstract

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.

Highlights

From the Departments of Neuroscience, Pharmucologyand Molecular Sciences,and Psychiatry, the Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the §Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Harvard Center for Kidney Diseases, Boston,Massachusetts 02115
Rabbits were immunized with IP3-sensitive store (IP3R) purified from rat cerebellum, and antibodies were purified on an IP3R affinity chromatography column
Substantial levels of IPBR distributed intothe nuclear pellet (Fig. 2 A, lane 3 ). This crude (800 X g) pellet contains 80% of the plasma membrane because, under the homogenization conditions used, the plasma membrane tends to sediment as large sheets. In this pellet is nearly half the rough endoplasmic reticulum (RER), which is contiguous with the nuclear envelope

Summary

Introduction

From the Departments of Neuroscience, Pharmucologyand Molecular Sciences,and Psychiatry, the Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the §Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Harvard Center for Kidney Diseases, Boston,Massachusetts 02115. Since about two-thirds of the RER is lostin the nuclear and mitochondrial pellets (panel B, lanes 3 and 5, respectively, and Ref. 27), the amountof IP3Ractually present inthe RER of canine pancreas is likely to be three times the amount evident in panel A ( l a n e 9).unlike ribophorin I (an RER-specific marker), the IP3R is presentinsubstantial quantities in both smooth microsomes (which contain mltnouc micro plasma membrane, as discussed below) and RM (highly purified RER), as well as a cushion fraction of intermediate density.

Results

Conclusion