Abstract
We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP(3)R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP(3)R. DANGER interacts with IP(3)R in vitro and co-immunoprecipitates with IP(3)R from cellular preparations. DANGER robustly enhances Ca(2+)-mediated inhibition of IP(3) RCa(2+) release without affecting IP(3) binding in microsomal assays and inhibits gating in single-channel recordings of IP(3)R. DANGER appears to allosterically modulate the sensitivity of IP(3) RtoCa(2+) inhibition, which likely alters IP(3)R-mediated Ca(2+) dynamics in cells where DANGER and IP(3)R are co-expressed.
Highlights
DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R
We have identified a novel vertebrate protein, designated DANGER, which was isolated by yeast two-hybrid analysis with the regulatory region of the IP3R as bait
A novel interacting sequence, corresponding to a previously uncharacterized expressed sequence tag designated KIAA1754 [7], bound to aa 923–1581 fragment of IP3R. This expressed sequence tag, which we denote as DANGER, maps to a human genomic sequence on chromosome 10q25.1 comprising a single coding exon (4.3 kb)
Summary
Right: GST pulldown assays using PC12 cell lysates (100 g) blotted with mouse monoclonal (M18A10) anti-type 1 IP3R antibody or polyclonal anti-type 3 IP3R antibody demonstrates binding to purified DANGER. D, co-immunoprecipitation experiments from (100 g) PC12 cell lysates of endogenous DANGER and type 1 IP3R, visualized by Western analysis. We observe robust interactions between a GST-DANGER fusion protein and IP3R (type 1 and type 3) from cell lysates as well as purified type 1 IP3R from rat cerebellum (Fig. 1B).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.