Subunit oligomerization, and topology of the inositol 1,4,5-trisphosphate receptor

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assemb ly of highly conserved subunits that contain multiple membrane-spanning seq uences in the C-terminal region of the protein. In studies aimed at investi gating the oligomerization and transmembrane topology of the type-1 InsP(3) R, a series of membrane-spanning region truncation and deletion plasmids we re constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to ass emble into tetrameric structures. The topology of the membrane-spanning reg ion truncations and the full-length receptor was determined by immunocytoch emical analysis of transfected COS-1 cells using complete or selective perm eabilization strategies, Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consisten t with the current model for the organization of the InsP(3)R in the endopl asmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenab le reagents to study receptor subunit oligomerization. Fractionation of sol uble and membrane protein components revealed that the first two membrane-s panning regions were necessary for membrane targeting of the receptor, Sedi mentation and immunoprecipitation experiments show that assembly of the rec eptor subunits was an additive process as the number of membrane-spanning r egions increased. Immunoprecipitations from cells co-expressing the full-le ngth receptor and carboxyl-terminal truncations reveal that constructs expr essing the first two or more membrane-spanning domains were capable of co-a ssembling with the full-length receptor. Inclusion of the fifth membrane-sp anning segment significantly enhanced the degree of oligomerization, Furthe rmore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membr ane-spanning region deletion constructions that terminate with the receptor 's 145 carboxyl-terminal amino acids were found to have enhanced assembly c haracteristics and implicate the carboxyl terminus as a determinant in olig omerization. Our results reveal a process of receptor assembly involving se veral distinct yet additive components and define the fifth and sixth membr ane spanning regions as the key determinants in receptor oligomerization.