title: Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor.
abstract: The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP(3)R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP(3)R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.
authors.affiliation: Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Galvan
authors.forename: E
authors.initials: E
authors.lastname: Borrego-Diaz
authors.forename: P J
authors.initials: PJ
authors.lastname: Perez
authors.forename: G A
authors.initials: GA
authors.lastname: Mignery
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D013301:Streptolysins / Q000494:pharmacology; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D013301:Streptolysins; C025964:streptolysin O