Structure and expression of the rat inositol 1,4,5-trisphosphate receptor.
The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.
Mignery
G A
GA
Department of Molecular Genetics, University of Texas Southwestern Medical School, Dallas 75235.
Newton
C L
CL
Archer
B T
BT
Südhof
T C
TC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000502:physiology; D015220:Calcium Channels; D002417:Cattle; D002531:Cerebellum / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012326:RNA Splicing; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000648:ultrastructure; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility; D014162:Transfection
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA