Expression of full-length and truncated recombinant human brain type I inositol 1,4,5-trisphosphate receptors in mammalian and insect cells.
Intracellular inositol 1,4,5-trisphosphate receptors (IP(3)Rs) form tetrameric Ca2+-release channels that are crucial for Ca2+ signalling in many eukaryotic cells. IP(3)R subunits contain an N-terminal, cytoplasmic, ligand binding domain linked by a modulatory domain to a channel-forming, hydrophobic C-terminal domain. We assembled and sequenced cDNAs encoding the SI-/SII+/SIII+ splice variant of the human brain type I IP(3)R, and functionally expressed the full-length receptor, and a C-terminally truncated receptor lacking the final 20% of the protein, in mammalian and insect cells. Both proteins were insoluble, consistent with in vivo immunofluorescence and ligand binding studies. This contrasted with the behaviour of recombinant FIKBP12 (a soluble control protein). The truncated receptor also fractionated with the "membrane" pellet after alkaline carbonate treatment. We conclude that the human type I IP(3)R forms high MW aggregates or complexes in cells when expressed without the C-terminal hydrophobic domain. This behaviour should be considered when expressing and refolding "soluble" human type I IP(3)R domains for structural studies.
Department of Biomedical Sciences, University of Edinburgh, United Kingdom.
T K
TK
Niu
R H
RH
Ashley
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D020104:Immunophilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007313:Insecta / Q000166:cytology / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D012995:Solubility; D022021:Tacrolimus Binding Proteins; D014162:Transfection
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins