title: Binding of thyroglobulin (Tg) to the low-density lipoprotein receptor-associated protein (RAP) during the biosynthetic pathway prevents premature Tg interactions with sortilin.
abstract: OBJECTIVE
  Sortilin, a Vps10p family member, is expressed by thyroid epithelial cells (TEC), where it binds to internalized thyroglobulin (Tg) molecules. Premature binding of Tg to sortilin during biosynthesis may cause intracellular retention of Tg. Such a premature interaction may be prevented by one or more inhibitor/s. Because both sortilin and Tg bind to the low-density lipoprotein receptor-associated protein (RAP), we investigated whether RAP serves such a function.
  
  
  METHODS
  Immunofluorescence staining for sortilin, Tg, and RAP was performed in FRTL-5 cells. Co-immunoprecipitation experiments were performed in extracts from FRTL-5 or COS-7 cells, the former co-transfected with Tg and/or RAP and/or sortilin, or in thyroid extracts from RAP KO mice.
  
  
  RESULTS
  Tg and sortilin did not co-localize in FRTL-5 cells following inhibition of protein synthesis, suggesting that newly synthesized, endogenous sortilin and Tg do not interact, in confirmation of which an anti-sortilin antibody did not co-precipitate Tg in FRTL-5 cells. In contrast, Tg co-localized with RAP in FRTL-5 cells. Co-immunoprecipitation of Tg with an anti-sortilin antibody in COS-7 cells transfected with sortilin and Tg was abolished when cells were co-transfected with RAP, indicating that RAP prevents binding of Tg to sortilin during biosynthesis, in confirmation of which an anti-sortilin antibody co-precipitated Tg in thyroid extracts from RAP KO mice to a greater extent than in thyroid extracts from WT mice.
  
  
  CONCLUSIONS
  Tg does not bind prematurely to sortilin because of its interaction with RAP during protein biosynthesis. These findings add new information to the knowledge of thyroid physiology.
authors.affiliation: Department of Clinical and Experimental Medicine, Endocrinology Units, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy.|DiaSorin S.p.A, Saluggia, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Botta
authors.affiliation: Department of Clinical and Experimental Medicine, Endocrinology Units, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy.|Neurobiology Laboratory of Biology, Scuola Normale Superiore, Piazza Dei Cavalieri 1, Pisa, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Lisi
authors.affiliation: Department of Clinical and Experimental Medicine, Endocrinology Units, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Rotondo Dottore
authors.affiliation: Department of Clinical and Experimental Medicine, Endocrinology Units, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Vitti
authors.affiliation: Department of Clinical and Experimental Medicine, Endocrinology Units, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy. michele.marino@med.unipi.it.
authors.forename: M
authors.initials: M
authors.lastname: Marinò
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D053898:Biosynthetic Pathways / Q000502:physiology; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D005260:Female; D026563:LDL-Receptor Related Protein-Associated Protein / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D013954:Thyroglobulin / Q000032:analysis / Q000378:metabolism*; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D026563:LDL-Receptor Related Protein-Associated Protein; D013954:Thyroglobulin; D000099183:Sortilin
keywords: Low-density lipoprotein receptor associated protein; RAP; Sortilin; Thyroglobulin; Thyroid