Over the last decade, there has been substantial improvement in the knowledge
of the cellular and molecular mechanisms governing the first steps of the thyroid
hormone secretory pathway, i.e. the internalization or endocytosis and intracellular
transport of the prohormone, thyroglobulin (Tg). Advances lie in the progressive
disappearance of dogmas and the input of convincing data either invalidating or
supporting rather old hypotheses.
Thyroid hormone production from the precursor glycoprotein, Tg is based on the
morpho-functional organization of polarized epithelial thyroid cells into follicle
structures delimiting an internal compartment, the follicle lumen. Tg molecules
secreted by thyrocytes into the lumen of thyroid follicles undergo, at the thyrocyte-lumen
interface, unique post-translational modifications (iodination and iodotyrosyl
residue coupling reactions) leading to the formation of hormone residues within
their polypeptide chains.
Depending on numerous factors - including the supply of iodide as substrate, the
activity of enzymes (thyroid peroxidase, TPO, and thyroid oxidase, ThOX) catalyzing
hormone formation, the concentration and physico-chemical state of Tg - the hormone
content of lumenal Tg molecules varies to a rather large extent. Tg molecules
newly arrived in the follicle lumen with no or a low hormone content would co-exist
with "older" Tg exhibiting up to 6-8 hormone residues. The downstream
processes responsible for the production of free thyroid hormones from these prohormonal
molecules must adequately manage the use of these lumenal heterogeneous Tg stores
to provide appropriate amounts of hormones for peripheral utilization. One would
expect to find control systems preventing excess hormone production that would
result from the processing of excessive amounts of prohormonal Tg molecules and
checking systems avoiding the use of Tg molecules with no or a low hormone content.
The way the thyroid follicle proceeds to generate free hormones from stored hormone
containing Tg molecules has been known for a long time. Tg molecules are taken
up by polarized thyrocytes and then conveyed to lysosomal compartments for proteolytic
changes that release T4 and T3 from their peptide linkages. There is an abundant
literature on these two steps, especially on the first one, which represents the
limiting point in the thyroid hormone secretory pathway. By contrast, the final
step, i.e. the "transfer" of free T4 and free T3 from the intracellular
compartments, in which they are generated, to the extracellular space, has never
been really studied and at present, there is no satisfactory explanation for the
transmembrane passage of these molecules that exhibit hydrophobic but also hydrophilic
characteristics owing to their head and tail (alanine side chain) charges.
The recent evolution in the knowledge of Tg endocytosis has first been to consider
that it could proceed via a mechanism different from phagocytosis ,also named
macropinocytosis, evidenced in rats under acute TSH stimulation (reviewed in 1).
Results from studies performed in rats have been for a long time and are still
in some instances extrapolated to the different animal species. Cellular events
characterizing macropinocytosis i.e. apical membrane extensions or pseudopods
and resorption vacuoles or colloid droplets are not or rarely observed in species
other than rat. There is now a number of experimental data showing that in the
thyroid of different species, internalization of Tg, mainly if not exclusively,
occurs via vesicle-mediated endocytosis or micropinocytosis (reviewed in 2),an
ubiquitous cellular process accounting for macromolecule internalization by all
A tentative explanation for the implication of macropinocytosis in rats
We have compared the Tg utilization rates required for a normal T4 production
in rats and human to try to find an explanation for the involvment of phagocytosis
/ macropinocytosis in Tg endocytosis in rats. Considering that the T4 production
rate is 85mg/day/15g thyroid tissue in human and about 3λg/day/15 mg tissue
for a rat weighing 250g, it appears that the production rate of T4 in λg/day/g
tissue is about 5 in human but 200 in rats. As under normal iodide supply, the
average hormone content of Tg should be similar in both species (about 2 T4
residues/molecule), it results that the amount of Tg degraded per day per g
tissue should be close to 2 mg in human and 40-50 times higher in rats.
From the general literature on endocytic vesicles and data deriving from studies
of thyroid micropinocytosis, one can estimate that endocytic vesicles with an
internal diameter of 50nm would contain at least 10 Tg molecules if, as very
likely (see below), Tg molecules enter these vesicles at a concentration of
about 200 mg/ml, representing the intralumenal Tg concentration. Using the above
mentioned values for the amount of Tg processed per day per g. tissue and assuming
that the rat and human thyroids are composed of about 107 (3) and
1010 cells respectively, we can calculate that, in the human gland,
the internalization of Tg would require a flux of about 200 vesicles per cell
per min.This value is fully compatible with the established values in cell types
as different as polarized kidney cells (4) or fibroblasts (5) which can form
up to 1000 vesicles per min.. In contrast, the vesicle flux that would be needed
in rats overrun by far this value. Thus, the vesicle-mediated endocytic process,
probably also operative in rats, would not have the capacity to internalize
Tg in sufficient amounts to sustain the T4 production required for homeostasis
in rats. Although speculative, these calculations give groundings for the implication,
in the rat, of an additional endocytic process with a high capacity such as
Steps and cellular compartments involved in Tg endocytosis by micropinocytosis
The internalization process starts with the organization of microdomains at
the apical plasma membrane of thyrocytes ; these microdomains or pits, resulting
from the recruitment and assembly of proteins (clathrin , adaptins
the cytoplasmic side of the membrane, invaginate to finally generate coated
vesicles after membrane fission. Lumenal Tg molecules, either free or associated
to membrane proteins acting as Tg receptors, enter the pits and are then sequestrated
into the newly-formed vesicles (6-8). The vesicles lose their coat and, through
a complex fusion process, deliver their content into a first type of endocytic
compartments, the early apical endosomes (see Figure). In these compartments,
Tg molecules probably undergo sorting on the basis of recognition of different
physico-chemical parameters either linked or independent such as the hormone
content, exposed carbohydrates, conformation of peptide domains
of sorting appears as a prerequisite for subsequent differential cellular handling
of Tg molecules. Indeed, it has been shown that internalized Tg molecules can
follow different intracellular pathways. Part of Tg molecules are conveyed via
a vesicle transport system to the second type of endocytic compartments,
late endosomes or prelysosomes (see Figure).
|Endocytosis and Transcytosis in the Thyroid Follicle.
|IA : Early apical endosome
||1-Initial phase of endocytosis
|IB : Early basolateral endosome
||2-Late phase of endocytosis
|II : Late endosome
|III : Lysosome
||a: processes initiated at the apical membrane
||b: processes initiated at the basolateral membrane
This route ending to lysosomes corresponds to the Tg degradation pathway for
the generation of free thyroid hormones. It is reasonable to think that Tg molecules
following this route are the more mature molecules (with a high hormone content)
but, this has not been firmly demonstrated. The other Tg molecules present in
early apical endosomes enter either of the following two routes; they are recycled
back into the follicle lumen through a direct vesicular transport towards the
apical plasma membrane (9) or via a two-step vesicular transport to the Golgi
apparatus and then to the apical plasma membrane (10). Alternately, Tg molecules
are transported and released at the basolateral membrane domain of thyrocytes
via transcytotic vesicles (11); a process accounting for the presence of Tg
in plasma (12-14). The orientation of Tg molecules towards one or the other
of these three routes requires the presence of receptors. However, at least
one intracellular pathway could simply convey Tg molecules that are not selected
for entering other routes.
Receptors involved in Tg endocytosis
Receptors may operate at the apical plasma membrane for Tg internalization and
downstream in apical early endosomes for Tg sorting. The requirement and/or
the involvement of apical cell surface receptors has long been debated. Most
investigators now recognize that receptors are not needed for internalization
since Tg is present at a high concentration at the site of vesicle formation.
So, Tg molecules are most likely internalized by fluid-phase endocytosis and
not by receptor-mediated endocytosis. On the contrary, if apical membrane Tg
receptors exist, their function would be to prevent the internalization of sub-classes
of Tg molecules (15,16). As it is not conceivable that internalized Tg molecules
could enter the different intracellular routes, described above, at random,
Tg receptors must exist in early apical endosomes. A detailed review on potential
Tg receptors has recently been made by Marino and Mc Cluskey (2).
The first candidate receptor, initially described by Consiglio et al.(17,18),
was later identified as the asialoglycoprotein receptor composed of three subunits
(RLH1,2 and 3). This receptor binds Tg at acidic pH and recognizes both sugar
moities and peptide determinants on Tg (19). As low-iodinated Tg molecules are
known to have a low sialic acid content, this receptor could be involved in
sorting immature Tg molecules for recycling to the follicle lumen. A second
receptor ,still not identified, named N-acetylglucosamine receptor (20), presumably
located in sub-apical compartments, interacts with Tg at acidic pH; it could
also act as a receptor for recycling immature Tg molecules back to the follicle
lumen. A third receptor; Megalin, has recently been discovered in the thyroid
and has been the subject of extensive studies yielding convincing data(2,21-23).
Megalin is an ubiquitous membrane protein belonging to the LDL receptor family.
It is located in the apical region of thyrocytes and its expression is regulated
by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with
a high affinity.In vitro and in vivo data indicate that Megalin is involved
in the transcellular transport or transcytosis of Tg molecules.
From the properties and subcellular location of these receptors, one can propose
an integrated view of the sorting processes that would operate in early apical
endosomes. The asialoglycoprotein receptor and the less defined N-acetylglucosamine
receptor would recognize immature Tg for recycling and Megalin would interact
with Tg subjected to apical to basolateral transcytosis. The remaining Tg molecules
would enter, without sorting, the functionally important pathway i.e. the prelysosome-lysosome
Connections between Apical and Basolateral Endocytosis
The capacity of thyrocytes to internalize macromolecules is not restricted
to the apical plasma membrane domain. Indeed, endocytosis of different proteins
including Tg and serum albumin also occurs at the basolateral plasma membrane
domain. Internalized molecules first enter early endocytic compartments, the
basolateral early endosomes (24). It has been demonstrated that internalized
proteins either reach late endosomes and lysosomes or undergo a transcellular
transport into the follicle lumen by basolateral to apical transcytosis. As
found in other cell types, thyroid late endosomes correspond to compartments
connecting apical and basolateral endocytic pathways. Basolateral endocytosis
and basolateral to apical transcytosis represent the route of entry of extrathyroidal
proteins such as plasma proteins found into the lumen of thyroid follicles (25).
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