S. Siffroi-fernandez et al., Identification of thyroglobulin domain(s) involved in cell-surface bindingand endocytosis, J ENDOCR, 170(1), 2001, pp. 217-226
Thyroglobulin (Tg) binds to cell surfaces through various binding sites of
high, moderate and low affinity. We have previously shown that binding with
low to moderate affinity is pH dependent, selective, but not tissue specif
ic. To identify the regions of Tg involved in this cell surface binding, we
studied the binding of I-125-labeled cyanogen bromide peptides from human
Tg to cell. surfaces of thyroid cells (inside-out follicles) and of CHO cel
ls. Electrophoretic analysis of cell homogenates after binding of native or
of reduced and alkylated I-125-labeled peptides showed that three peptides
, P1, P2 and P3, were always associated with the cells. Sequence analysis a
llowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (
Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among
which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 t
o Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly
(P2 and P3-2) located in the C-terminal domain of Tg homologous With acetyl
cholinesterase. The smallest peptides, P1 and P2, were purified by preparat
ive electrophoresis. They both displayed strong binding properties towards
cell surfaces. Inhibition experiments of I-125-labeled Tg binding by P1 or
P2 indicated that they were involved in Tg binding to cell surfaces. All th
e other peptides tested for their binding abilities were either not or only
poorly involved in Tg binding to cell surfaces, which suggested that P1 an
d P2 are major Tg sites of binding to cell surfaces. These two peptides are
not involved in the binding of Tg to the known Tg 'receptors' described in
the literature, to which recycling, transcytosis and regulation functions
have been ascribed. Thus they are potential tools to identify cell surface
components involved in the process of Tg endocytosis leading to lysosomal d
egradation.