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

摘要

pIn an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate [Ins(1,4,5)iP/isub3/sub] receptor [Ins(1,4,5)iP/isub3/subR] 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)iP/isub3/subR 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)iP/isub3/subR(ES) fusion protein in iXenopus laevis/ioocytes, 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)iP/isub3/subR(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous iXenopus/i Ins(1,4,5)iP/isub3/subR shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)iP/isub3/subR(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)iP/isub3/subR(ES) constitutes the membrane spanning domain of the Ins(1,4,5)iP/isub3/subR 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)iP/isub3/subR(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor./p