Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum

Abstract

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca(2+) from the ER elicited by thapsigargin (a specific inhibitor of ER Ca(2+)-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca(2+) strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

The Acetylcholinesterase Homology Region Is Essential for Normal Conformational Maturation and Secretion of Thyroglobulin

“…7A). As expected, the mobility of monomeric 330-kDa bTg or bTg⅐hAChE is faster under nonreducing conditions because of the presence of the many intrachain disulfide bonds (39). More importantly, under nonreducing conditions, a disulfide-linked bTg⅐hAChE dimer band appeared (Fig.…”

TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2+-ATPases expression and activity in PC Cl3 thyroid cells

“…It is well known that protein folding in the ER requires a high Ca 2þ concentration (3). Moreover, we have directly demonstrated a Ca 2þ requirement for the folding of Tg (17)(18)(19). On this basis, it could be expected that an increased ER protein load would correspond to an increased capability to take up Ca 2þ in the ER lumen, i.e.…”

“…However, there is a scarcity of data concerning the regulation of SERCA2b. Several SERCA-modulated cellular functions in the thyroid have been studied by our group (13)(14)(15)(16)(17)(18)(19). Very recently, we reported that SERCA2b is the main SERCA form expressed in thyroid and that SERCA2b expression is down-regulated by neoplastic transformation of the thyroid cell line PC Cl3 (20).…”

Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway

“…However, ER stress also plays an important role in the beta cell failure that precipitates type 2 diabetes. Beta cells, like plasma cells and thyroid cells, have a high protein load [32,33], synthesising large quantities of (a single) protein. Proinsulin represents up to 20% of the total mRNA and 30-50% of the total protein synthesis of the beta cell [18,19].…”