Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway.
Secretion of thyroglobulin (Tg) by thyrocytes requires several endoplasmic reticulum (ER)-resident molecular chaperones. The receptor-associated protein (RAP), a known molecular chaperone, binds to Tg in thyroid cells shortly after biosynthesis. Here we investigated whether RAP is involved in Tg secretion by FRTL-5 cells. For this purpose, we studied Tg secretion by FRTL-5 cells transfected with a soluble RAP chimera, as a mean for interfering with endogenous RAP. We used a RAP-human IgG Fc (RAP-Ig) chimeric cDNA, which was designed in order to exclude the ER retention sequence of RAP and to allow generation of a secreted form of RAP. FRTL-5 cells were transiently transfected with the RAP-Ig cDNA or, as control, with a CD8-Ig cDNA. Media were collected at 24, 48 and 72 h after transfection. Secretion of fusion proteins and of Tg in the media was measured by ELISA. As expected, under standard culture conditions, RAP was not secreted into the media by FRTL-5 cells, even though it could be detected by Western blotting in cell extracts. In transfection experiments, fusion proteins were present in the media of FRTL-5 cells transfected with either RAP-Ig or CD8-Ig, indicating that transfection was successful. Although Tg was found in the media of FRTL-5 cells transfected with either CD8-Ig or RAP-Ig, a lower amount was found in cells transfected with RAP-Ig. Therefore, we concluded that RAP is involved in Tg secretion by FRTL-5 cells suggesting that RAP may function as a Tg molecular chaperone.
Department of Endocrinology, University of Pisa, Pisa, Italy.
S
S
Lisi
L
L
Chiovato
A
A
Pinchera
C
C
Marcocci
F
F
Menconi
E
E
Morabito
M A
MA
Altea
R T
RT
McCluskey
M
M
Marinò
D015153:Blotting, Western; D016827:CD8 Antigens / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013954:Thyroglobulin / Q000096:biosynthesis / Q000378:metabolism*; D013961:Thyroid Gland / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D016827:CD8 Antigens; D026563:LDL-Receptor Related Protein-Associated Protein; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D013954:Thyroglobulin