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

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.