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 photoaffinity labeling with [125I]T4 to possess a single L-T4 binding site with a K5 of about 3 x 10(7) M-1 and a relative affinity for analogs of L-T4 = D-T4 = rT3 = triiodothyroacetic 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 site 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 15- to 26-kilodalton (kDa) fragments (within residues 1-160 to 210), but not the approximately 10- to 11-kDa fragments (within residues approximately 220-299), were labeled by [125I]T4. 2) Variants apoE2 and apoE4, with nonconservative mutations at positions 112 and 158 (the latter unable to interact with the apoB/E receptor), maintained the ability to bind T4. 3) Monoclonal antibodies MAb 1D7 and 3H1 (epitopes at positions 139-169 and 243-272, respectively) failed to inhibit T4 binding, but MAb 6H7 (epitope at 1-125) decreased labeling by about 24%. 4) Polymers of apoE were specifically labeled despite the interaction between amphipathic alpha-helices of the exon 4-encoded region (63-299). We conclude that apoE, as previously observed with apoA-I and apoB, possesses 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 by cells through apoB/E receptors, which are widely distributed in tissues.
Benvenga
S
S
Cattedra di Endocrinologia, University of Messina School of Medicine, Italy.
Cahnmann
H J
HJ
Robbins
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D004008:Diclofenac / Q000494:pharmacology; D005091:Exons; D005665:Furosemide / Q000494:pharmacology; D007525:Isoelectric Focusing; D008528:Mefenamic Acid / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D013974:Thyroxine / Q000378:metabolism*
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; D001798:Blood Proteins; D004008:Diclofenac; D008528:Mefenamic Acid; D005665:Furosemide; D013974:Thyroxine