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Direct association of ligand‐binding and pore domains in homo‐ and heterotetrameric inositol 1,4,5‐trisphosphate receptors
Author(s) -
Boehning Darren,
Joseph Suresh K.
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.20.5450
Subject(s) - biology , protein subunit , c terminus , gene isoform , n terminus , fusion protein , immunoprecipitation , inositol , receptor , biochemistry , ligand (biochemistry) , microbiology and biotechnology , binding site , biophysics , peptide sequence , recombinant dna , amino acid , gene
Inositol 1,4,5‐trisphosphate receptors (IP 3 Rs) are a family of intracellular Ca 2+ channels that exist as homo‐ or heterotetramers. In order to determine whether the N‐terminal ligand‐binding domain is in close physical proximity to the C‐terminal pore domain, we prepared microsomal membranes from COS‐7 cells expressing recombinant type I and type III IP 3 R isoforms. Trypsin digestion followed by cross‐linking and co‐immunoprecipitation of peptide fragments suggested an inter‐subunit N‐ and C‐terminal interaction in both homo‐ and heterotetramers. This observation was further supported by the ability of in vitro translated C‐terminal peptides to interact specifically with an N‐terminal fusion protein. Using a 45 Ca 2+ flux assay, we provide functional evidence that the ligand‐binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP 3 Rs and propose that the gating mechanism of IP 3 R Ca 2+ channels involves the association of the N‐terminus of one subunit with the C‐terminus of an adjacent subunit in both homo‐ and heterotetrameric complexes.