Endocrinology Vol. 145, No. 1 1-3
Copyright © 2004 by The Endocrine Society
A Physiological Role for the Posttranslational Cleavage of the Thyrotropin Receptor?
Gilbert Vassart and
Sabine Costagliola
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Faculté de Médecine, Université Libre de Bruxelles and Service de Génétique Médicale, Hôpital Erasme, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Gilbert Vassart, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Faculté de Médecine, Université Libre de Bruxelles and Service de Génétique Médicale, Hôpital Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: gvassart{at}ulb.ac.be.
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Introduction
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Together with the LH/chorionic gonadotropin receptor (LH/CGr) and FSH receptor (FSHr), the TSH receptor (TSHr) constitute the glycoprotein hormone receptor family, a subclass of rhodopsin-like G protein-coupled receptors (GPCRs). All three receptors contain a large extracellular amino terminal domain responsible for hormone recognition and binding, upstream of a serpentine portion with seven transmembrane helices typical of GPCRs. Contrary to the LH/CGr and FSHr, which are single-chain molecules, the TSHr was shown to be a heterodimer (1), even before its cloning in 1989 (2, 3, 4). After some initial controversies, the consensus was reached that a single-chain monomer was posttranslationally cleaved at the cell surface, by a still ill-defined metalloprotease (5, 6, 7). The resulting protomers, termed TSHr
or TSHR-A (for the amino terminal portion) and TSHrß or TSHR-B, respectively, are held together by disulfide bonds, and a segment of about 50 residues is removed from the amino terminal end of ß-subunit (7, 8).
This peculiarity of the TSHr has attracted much attention and a role for the cleavage of the TSHr has been looked for in (patho)physiology. The report that following cleavage of the single-chain precursor, a proportion of the resulting -subunit was released secondary to the action of protein disulfide isomerase (9) opened the additional question of the putative function(s) of the shedded -subunit, and of the beheaded ß-subunit remaining in the plasma membrane. We will briefly consider below some of the questions and current controversies related to the heterodimeric nature of the TSHr. The separate issue of whether heterodimeric TSHr molecules associate further into the membrane to form multimers, and the possible functional significance of this phenomenon, will not be considered here.
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What Is the Situation in the Thyrocyte in Vivo?
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Because there is currently no fool-proof method allowing to measure directly the proportion of single-chain and dimeric receptors, at the cell surface in intact thyroid tissue, we have no definite answer to this question. Available results suffer from the lack of internal controls for recovery of the various molecular species. From the early results of Rees-Smith and colleagues (1) and more recent data of several groups, the consensus emerges that the cleaved receptor is the prevalent molecular form in native tissue, whereas variable proportions of single-chain and cleaved receptors are found in cells transfected with recombinant receptors. Although logical, the notion that continuous shedding of the -subunit causes accumulation of a significant excess of truncated receptors made only of ß-subunits (5) must be taken with caution (see above). It must be kept in mind that shedding of -subunit has only be documented in cell culture (primocultured thyrocytes and cultured transfected cells) (9) and that no precise method currently allows quantification of the ß-subunit alone in intact tissue.
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A (Patho)Physiological Role for the Released -Subunit?
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It is tempting to draw a parallel between shedding of the hormone-binding domain of the TSHr and similar situations documented for receptors belonging to other gene families (e.g. GH or prolactin receptors, cytokine receptors, etc.) (10). In these cases, the hormone-binding domains released in the circulation by proteolysis (or sometimes, translated from alternatively spliced transcripts) have been shown to play the role of buffers or enhancers of hormone action, or transporters. The recent demonstration that high affinity binding of TSH to its receptor requires the presence of sulfated tyrosine residues, located in the ß-subunit (11), makes this role unlikely in the case of the TSHr. Whereas it readily explains the absence of hormone binding observed with purified -subunit constructs (12), it is more difficult to reconcile the need for sulfated tyrosines with results showing TSH binding to shedded -subunits (9). Suggestions that shedding of the -subunit may play a role in the pathogenesis of Graves’ disease may be more relevant. Indeed, it has been shown that the stimulating autoantibodies characteristic of this disease recognize more efficiently the amino terminal ectodomain of the receptor (essentially the -subunit) when it is not linked to the serpentine portion (13, 14, 15). We would like to propose the hypothesis that extrathyroidal manifestations of Graves’ disease might be related to metastasis of the autoimmune reaction to extrathyroidal sites via the released -subunit.
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The Isolated ß-Subunit Displays Constitutive Activity
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A series of experiments from several groups demonstrate clearly that, when expressed by transfection in nonthyroid cells, truncated TSHr constructs corresponding to ß-subunits display increase in constitutive activity (16, 17, 18). Such beheaded receptors are, however, far from fully activated, as they can be further stimulated by a selection of mutations found in autonomous adenomas (16, 18). In the absence of knowledge of the actual amount of excess ß-subunit present in the plasma membrane of thyrocytes at steady state, it is difficult to argue for (or against) a physiological role of the isolated ß-subunit. However, these observations, together with results obtained with the gonadotropin receptors (19, 20), have been extremely informative to approach the intramolecular mechanisms implicated in activation of the receptor. They lead us to propose that, upon binding of TSH, the ectodomain of the receptor would switch from an inverse agonist, keeping low the basal activity of the serpentine, into a full agonist of the serpentine portion (16). According to this model, activation of the TSHr would result from the interaction of the complex between the ectodomain and TSH with the serpentine portion of the receptor. Because point mutations in selected residues of the ectodomain can fully activate the receptor (21), it is tempting to conclude that the real immediate agonist of the serpentine portion of the receptor is an activated conformation of the ectodomain stabilized by TSH binding (16).
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A Physiological Role for the Cleavage Mechanism?
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The observation that the TSHrs at the surface of thyrocytes present (variable?) proportions of single-chain and cleaved, two-subunit molecules (see above), poses the question of a possible functional difference between the two forms. Experiments with noncleavable mutant constructs gave the clear-cut answer that these were functional, and that there was minimal effect if any on all measurable parameters (Ref. 7 and our unpublished results). Of course, these experiments, performed in transfected nonthyroidal cells, do not rule out a functional difference between cleaved and noncleaved receptors in the thyrocyte in vivo. They do not point to receptor cleavage, however, as an appealing regulatory step.
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Two Recent Reports Challenge the Above Picture
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In the first, using two different approaches, Ciullo et al. (22) concluded that only the cleaved two-chain TSHr is able to signal to the Gs protein. This contradicts the results obtained both with the truncated and noncleavable receptors (see above). The authors suggest that differences obtained with truncated mutants, which in their hands did not signal to Gs, might be due to their using stable, rather than transient, transfections. We suggest that low or absent expression of their truncated mutants at the cell surface would be an alternative explanation. We and others have shown that in the absence of a driver sequence engineered in the amino terminus of the constructs, the truncated mutants do not reach the cell surface (16, 17). Flow immunocytometry data of Ciullo et al. (22) showing the presence of the truncated receptor in only a small minority of their stable transfectants (which might be due to unwanted cell permeabilization) agree with this interpretation. Regarding the discrepancy of their results with the observation that noncleavable receptors do signal normally (see above), they proposed that the noncleavable constructs might not mimic the structure of wild-type uncleaved receptors. Experimental probing of this question may prove difficult. The protease inhibitor BB-2116, which has been shown to inhibit shedding of -subunit (9) could be used to this aim.
In the second, Ando et al. (23) proposed that activation of the TSHr by TSH, but not by a recently described stimulating monoclonal antibody (MS-1), causes cleavage of the single-chain TSHr. Despite the absence of solid evidence for a functional difference between single-chain and two-chain receptors (see above), these results, if confirmed, might indeed be a strong indication in favor of such a difference. In the present issue of Endocrinology, Chazenbalk et al. (24) have attempted replication of the results of Ando et al. under identical experimental conditions, but with different methodologies. The results were clearly negative: in their hands, if anything, TSH inhibits slightly cleavage of the single-chain TSHr. Time and use of still other methodologies will tell who is right.
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Do We Need to Find a Role for Cleavage of TSHr?
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Implicit in the reasoning of most of us is the notion that there must be a function associated with any structural peculiarity of the molecules or systems we are studying. This is obviously a major driving force underlying many detailed investigations, and the molecular dissection of the mechanisms implicated cleavage of the TSHr provides a good example of it. As all the phenomena or structures we are studying in biology are the result of evolution, we should not forget, however, that genetic drift or neutral evolution challenges this notion. One way to explore the role of TSHr cleavage experimentally would be to generate knock-in mice with noncleavable receptor mutants and to study their phenotype in detail. The future will tell whether the driving force is strong enough to overcome the energy barrier associated with such project.
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Footnotes
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Abbreviations: FSHr, FSH receptor; GPCR, G protein-coupled receptor; LH/CGr, LH/chorionic gonadotropin receptor; TSHr, TSH receptor.
Received September 15, 2003.
Accepted for publication September 22, 2003.
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Introduction
What Is the Situation...