Factors Determining the Composition of Inositol Trisphosphate Receptor Hetero-oligomers Expressed in COS Cells

Abstract

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 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 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.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

“…Briefly, to make the head subunit, IP 3 R2 cDNA in pcDNA3.1(ϩ) vector was modified so that all NcoI sites in the coding region were silently mutated (primers [15][16][17][18]. A glycine codon (GGC) was inserted after the initiation methionine to create a new NcoI site and a Kozak sequence in the context of the start codon (primer 19).…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…The InsP 3 Rs are ϳ2,700 -2,800 amino acid intracellular membrane proteins that exist as homo-or heterotetramers (209,210,212,270,311,328,363,443,536). By analogy with other cation channels and some structural information, the ion-conducting pore is believed to be created at the central axis of the tetrameric structure (Fig.…”

Carboxyl-terminal Sequences Critical for Inositol 1,4,5-Trisphosphate Receptor Subunit Assembly

“…Though heterotetramer formation between the transfected and endogenous receptor populations are possible, the vast majority of the overexpressed constructs are homotetrameric assemblies of the transfected template (17,24). Joseph et al (25) suggested that formation of the heteromeric complexes was dependent upon relative expression of the two isoforms and that there may be kinetic constraints that favor homo-oligomers over hetero-oligomers.…”