The cholinesterase-like domain, essential in thyroglobulin trafficking for thyroid hormone synthesis, is required for protein dimerization.
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted alpha-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.
Lee
Jaemin
J
Cell and Molecular Biology Program, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Wang
Xiaofan
X
Di Jeso
Bruno
B
Arvan
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002802:Cholinesterases / Q000737:chemistry*; D000939:Epitopes; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013954:Thyroglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013963:Thyroid Hormones / Q000096:biosynthesis*
D000939:Epitopes; D013963:Thyroid Hormones; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013954:Thyroglobulin; D002802:Cholinesterases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase