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J. Biol. Chem., Vol. 275, Issue 21, 16084-16090, May 26, 2000
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From the Department of Pathology and Cell Biology, Thomas Jefferson
University School of Medicine, Philadelphia, Pennsylvania 19107
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 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 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.
Factors Determining the Composition of Inositol Trisphosphate
Receptor Hetero-oligomers Expressed in COS Cells*
,
*
This work was supported by National Institutes of Health
Grants T32-AA07463 (to S. B and D. B.), RO1-GM58574, and R01-AA10971.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology and
Cell Biology, Thomas Jefferson University, Rm. 230A JAH, 1020 Locust
St., Philadelphia, PA 19107. Tel.: 215-503-1221; Fax: 215-923-6813;
E-mail: Suresh.Joseph@mail.tju.edu.
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