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

In an attempt to define structural regions of the type I inositol 1,4, 5-trisphosphate [Ins(1,4,5)P-3] receptor [Ins(1,4,5)P(3)R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we hav e employed the use of green fluorescent protein (GFP) to monitor the l ocalization of a truncated Ins(1,4,5)P(3)R 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)P(3)R(ES) fusion protein i n Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microsc opy 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)P(3)R(ES) to perinuclea r ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4,5)P(3 )R shown previously. Furthermore, electron microscopy data indicate th at expressed GFP-Ins(1,4,5)P(3)R(ES) is in fact targeted to the ER. So dium carbonate extraction of microsomal membranes and cross-linking ex periments indicate that the expressed chimeric protein is in fact memb rane anchored and able to form a homotetrameric complex. Our data prov ides evidence that Ins(1,4,5)P(3)R(ES) constitutes the membrane spanni ng domain of the Ins(1,4,5)P(3)R and is able to mediate homotetramer f ormation, without the need for the large N-terminal cytoplasmic domain . Furthermore, the localization of GFP-Ins(1,4,5)P(3)R(ES) on the ER i ndicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.