Trypsin digestion of the inositol trisphosphate receptor: implications for the conformation and domain organization of the protein.
Limited digestion of rat cerebellum microsomal vesicles with trypsin resulted in the proteolysis of the 240 kDa inositol 1,4,5-trisphosphate receptor (IP3R) and the formation of a 94 kDa species that remained membrane-bound and retained immunoreactivity to an antibody raised against the C-terminal sequence of this protein. The appearance of the 94 kDa species was associated with a loss of [3H]IP3 binding sites in the membrane and the appearance of [3H]IP3 binding sites in the soluble fraction. The 94 kDa fragment retained reactivity to biotinylated concanavalin A. In vitro phosphorylation of the IP3R in membranes with cyclic AMP-dependent protein kinase and [gamma-32P]ATP produced an unlabelled 94 kDa fragment after tryptic digestion. According to current models of the cerebellar IP3R this would place the proteolytic site between the phosphorylation site at serine-1755 and the first transmembrane segment of the IP3R. A second antibody raised to amino acids 401-414 in the N-terminal region of the receptor recognizes a 68 kDa fragment released into the soluble fraction after trypsin treatment. The time course of release of the 68 kDa fragment was correlated with the appearance of soluble binding sites, and the fragment was bound by IP3-Affigel resin. A large proportion of the 68 kDa fragment remained associated with the membrane fraction and could be specifically immunoprecipitated from detergent extracts of digested membranes by anti-C-terminus antibody. Our results provide experimental evidence to further localize the ligand binding domain and suggest that regions of the N-terminus and C-terminus may be non-covalently associated.
Joseph
S K
SK
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, PA 19107, USA.
Pierson
S
S
Samanta
S
S
D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism / Q000648:ultrastructure; D006031:Glycosylation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D012116:Resins, Plant; D012995:Solubility; D014316:Tritium; D014357:Trypsin / Q000378:metabolism* / Q000494:pharmacology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D012116:Resins, Plant; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D014357:Trypsin