Factors determining the composition of inositol trisphosphate receptor hetero-oligomers expressed in COS cells.
COS-7 cells were transiently transfected with type I and type III myo-inositol 1,4,5-trisphosphate receptor (IP(3)R) 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 IP(3)R to distinguish the transfected from the endogenous isoform. This was not necessary for the type I IP(3)R, since the endogenous levels of this isoform were extremely low. Based on sucrose gradient analysis, the transfected type I or FLAG-type III IP(3)Rs assembled into tetramers. Confocal immunofluorescence experiments confirmed that the constructs were primarily targeted to the endoplasmic reticulum. Recombinant type I IP(3)R expressed in COS cells over a 48-h period showed a negligible capacity to form hetero-oligomers with endogenous type III IP(3)Rs, based upon co-immunoprecipitation 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 IP(3)Rs. 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 IP(3)R 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 (35)S 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.
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Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA. Suresh.Joseph@mail.tju.edu
Bokkala
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Boehning
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D000818:Animals; D019556:COS Cells; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D009842:Oligopeptides; D010455:Peptides; D011233:Precipitin Tests; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014162:Transfection
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009842:Oligopeptides; D010455:Peptides; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C085706:FLAG peptide