COS-7 cells were transiently transfected with type I and type III myo- inositol 1,4,5-trisphosphate receptor (IP3R) isoforms to study the processes underlying assembly and oligomerization of these tetrameric proteins. A FLAG epitope was engineered on to the N terminus of the type III IP3R to distinguish the transfected from the endogenous isoform. This was not necessary for the type I IP3R, since the endogenous levels of this isoform were extremely low. Based on sucrose gradient analysis, the transfected type I or FLAG-type III IP3Rs assembled into tetramers. Confocal immunofluorescence experiments confirmed that the constructs were primarily targeted to the endoplasmic reticulum. Recombinant type I IP3R expressed in COS cells over a 48-h period showed a negligible capacity to form hetero- oligomers with endogenous type III IP3Rs, based upon coimmunoprecipitation assays. However, substantial formation of hetero-oligomers was observed between recombinant receptors when the cells were simultaneously transfected with type I and FLAG-type III IP3Rs. Co-immunoprecipitation experiments using lysates from metabolically labeled cells allowed the quantitation of homo- and hetero-oligomers in cells transfected with different ratios of type I and FLAG-type III IP3R DNA. These studies show that the relative expression level of the two isoforms influences the fraction of hetero- oligomers formed. However, the proportion of hetero-oligomers formed were less than predicted by a binomial model in which the association of subunits is assumed to be random. In doubly transfected cells, the early kinetics of 35S label incorporation into homotetramers showed a lag period corresponding to the time taken to synthesize a full-length receptor. However, hetero-oligomers were synthesized with a longer lag period, suggesting that there may be kinetic constraints that favor homo-oligomers over hetero-oligomers.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology