Transmembrane topology and sites of N-glycosylation of inositol 1,4,5-trisphosphate receptor.
To define the transmembrane topology of the inositol 1,4,5-trisphosphate receptor (InsP3R), we determined the subcellular location of the hydrophilic segment (residues 2463-2529 of mouse type 1 InsP3R) believed to be located at the luminal side of the endoplasmic reticulum (ER) in the six-transmembrane model but at the cytoplasmic side in the eight-transmembrane model. This hydrophilic segment includes two consensus sites for N-glycosylation (Asn-2475 and Asn-2503). We prepared an anti-peptide antibody against residues 2504-2523. Electron microscope immunocytochemical studies of mouse cerebellar Purkinje cells showed that binding of this antibody frequently occurs in the intracisternal space of the ER. We constructed three mutant receptors by site-directed mutagenesis of Asn to Gln (N2475Q, N2503Q, and N2475Q/N2503Q). By concanavalin A column chromatography of these receptors, we found that both Asn-2475 and Asn-2503 are glycosylated. These results indicate that residues 2504-2523, Asn-2475, and Asn-2503 are exposed to the ER lumen. We therefore propose that InsP3R has six membrane-spanning segments. Based on the transmembrane topology and subunit organization, we suggest that InsP3R is a member of the superfamily that includes the voltage- and second messenger-gated ion channels on the plasma membrane.
Michikawa
T
T
Department of Molecular Neurobiology, University of Tokyo, Japan.
Hamanaka
H
H
Otsu
H
H
Yamamoto
A
A
Miyawaki
A
A
Furuichi
T
T
Tashiro
Y
Y
Mikoshiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000648:ultrastructure*; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D006031:Glycosylation; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007425:Intracellular Membranes / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000648:ultrastructure*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018160:Receptors, Cytoplasmic and Nuclear / Q000648:ultrastructure*; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D003208:Concanavalin A; D015544:Inositol 1,4,5-Trisphosphate