Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors.
Inositol 1,4,5-trisphosphate (IP3) receptors are ligand-gated channels that release intracellular Ca2+ stores in response to the second messenger, IP3. We investigated the potential role of IP3 receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca2+ mobilization is required for nuclear vesicle fusion and implicated IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibited IP3-stimulated CA2+ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP3. Because nuclear vesicle fusion was inhibited by antibodies that block Ca2+ flux, but not by control and preimmune antibodies, we concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown.
Sullivan
K M
KM
Department of Cell Biology and Anatomy, Johns Hopkins University Schhol of Medicine, Baltimore, MD 21205, USA.
Lin
D D
DD
Agnew
W
W
Wilson
K L
KL
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects* / Q000276:immunology; D002474:Cell-Free System; D000939:Epitopes; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008561:Membrane Fusion / Q000187:drug effects*; D008938:Mitosis / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism*; D010063:Ovum; D011233:Precipitin Tests; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects* / Q000276:immunology; D014981:Xenopus
D000906:Antibodies; D015220:Calcium Channels; D000939:Epitopes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium