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Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.
Sayers LG, Miyawaki A, Muto A, Takeshita H, Yamamoto A, Michikawa T, Furuichi T, Mikoshiba K.
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In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.
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