CHARACTERIZATION OF THYROID-HORMONE BINDING TO APOLIPOPROTEIN-E - LOCALIZATION OF THE BINDING-SITE IN THE EXON 3-CODED DOMAIN

Apolipoprotein-E (apoE) has been shown by noncovalent binding and phot oaffinity labeling with [I-126]T4 to possess a single L-T, binding sit e with a K. of about 3 x 10(7) m-1 and a relative affinity for analogs of L-T4 = D-T4 = rT3 = triodothyroacetic acid > L-T3. T4 binding was not affected by the flavonoid EMD 21388 or heparin, but was inhibited by diclofenac = mefenamic acid > furosemide. Localization of the T4 si te to the N-terminal 62-amino acid region of the mature peptide coded by exon 3 was deduced from the following evidence. 1) The N-terminal 1 5- to 26-kilodalton (kDa) fragments (within residues 1-160 to 210), bu t not the approximately 10- to 11-kDa fragments (within residues appro ximately 220-299), were labeled by [I-125]T4. 2) Variants apoE2 and ap oE4, with nonconservative mutations at positions 112 and 158 (the latt er unable to interact with the apoB/E receptor), maintained the abilit y to bind T4. 3) Monoclonal antibodies MAb 1D7 and 3H1 (epitopes at po sitions 139-169 and 243-272, respectively) failed to inhibit T4 bindin g, but MAb 6H7 (epitope at 1-125) decreased labeling by about 24%. 4) Polymers of apoE were specifically labeled despite the interaction bet ween amphipathic alpha-helices of the exon 4-encoded region (63-299). We conclude that apoE, as previously observed with apoA-I and apoB, po ssesses a T4-binding domain separate from the lipid-binding domain and distinct from both the heparin- and the cell receptor-binding sites. Thyroid hormone binding by apoE may facilitate uptake of the hormone b y cells through apoB/E receptors, which are widely distributed in tiss ues.