Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2016 Apr;37(2):114-34.
doi: 10.1210/er.2015-1098. Epub 2016 Jan 22.

TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective

Basil Rapoport  1 , Sandra M McLachlan  1
Affiliations
Review

TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective

Basil Rapoport et al. Endocr Rev. 2016 Apr.

Retraction in

  • Erratum Notice for Duplicate Publications.
    [No authors listed] [No authors listed] Endocr Rev. 2020 Dec 1;41(6):886. doi: 10.1210/endrev/bnaa019. Endocr Rev. 2020. PMID: 32805736 Free PMC article. No abstract available.

Abstract

The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
A, Concept 30 years ago of TSHR intramolecular cleavage into subunits based on 125I-TSH cross-linking to FRTL5 thyroid cells before molecular cloning of the receptor (6). A single polypeptide chain with an extracellular disulfide-bonded loop was proposed to undergo intramolecular proteolysis with removal of a portion of the loop resulting in two disulfide-linked subunits. Experimentally, these subunits could be separated by reduction of these disulfide bonds. Note the terminology of A- and B-subunits. [Reproduced from Figure 4 in J. Furmaniak et al: Photoaffinity labelling of the TSH receptor on FRTL5 cells. FEBS Lett. 1987;215:316–322 (6), with permission. © Elsevier.] B, Schematic representation of the TSHR as presently understood. Aside from more information on the molecular structure of the A- and B-subunits and the realization that there are seven transmembrane passages in the B-subunit, the basic concept is the same as that proposed 30 years ago. The three cysteine clusters in the TSHR ECD are indicated as I, II and III. The A- and B-subunits are depicted as separated by elimination of disulfide bonding between cysteine clusters II and III. The dotted arc indicates the approximate ligand binding site, in the case of TSH binding extending into the N-terminus of the B-subunit.
Figure 2.
Figure 2.
A, Chimeric TSH-LH receptors used to investigate TSHR intramolecular cleavage into subunits. Schematic representation of TSHR and LHR components are shown in red and blue, respectively. Only the combined substitution of TSHR domains D and E with the homologous regions of the LHR (chimera 6) abolished TSHR cleavage. Individual substitutions were without effect. Smaller individual chimeric or alanine substitutions throughout the TSHR (termed “mini” substitutions), as well as deletion of the approximately 50 amino acid residues (317–366) present in the TSHR and absent in the LHR, also failed to prevent receptor cleavage. However, the small chimeric substitution of TSHR residues 367–369 with the comparable region of the LHR (residues 317–319) together with deletion of TSHR residues 367–369 did abolish cleavage (TSHR D1-NET). This chimeric substitution eliminates an N-linked glycan (green circle) present in the LHR. B, Schematic representation of TSHR A- and B-subunit sizes depending on the theoretical or experimentally observed size of these subunits. The A-subunit sizes indicated are after enzymatic removal of N-linked glycan moieties. The deleted C-peptide region cannot be isolated experimentally, and therefore appears to be degraded after clipping at two (upstream and downstream) cleavage sites, or, more likely, initial clipping at site 1 site is followed by progressive “salami slicing” downstream to site 2. There is greater consensus regarding the approximate size of the A-subunit (C-terminus between residues 310–330) than for the B-subunit. A minority of B-subunits are near site 1 (hence “big”) and probably represent species formed directly after initial cleavage. The dominant B-subunit species have N-termini at 370 and 378.
Figure 3.
Figure 3.
Both the single-chain and cleaved, two-subunit TSHR expressed on the cell surface contain mature complex glycan moieties. Only single-chain TSHR with immature high mannose glycan moieties are present within the cell. In this representative experiment, TSHR stably expressed by Chinese hamster ovary cells were pulse-chased with radiolabeled amino acid precursors, and receptors present on the cell surface were separated from receptors within the cell, followed by disulfide bond reduction, enzymatic deglycosylation, and autoradiography (27). Endoglycosidase H (Endo H) removes only high mannose glycan, whereas endoglycosidase F (Endo F) removes both high mannose and complex glycan. For cell surface TSHR, single-chain and two-subunit receptors are present that are both resistant to Endo H but deglycosylated by Endo F. Note the very large glycan contribution to the A-subunits, with a post deglycosylation reduction in size from approximately 60 kDa to approximately 33 kDa. The B-subunit lacks glycan moieties and appears as a “smear” extending from approximately 44–40 kDa, indicating a greater susceptibility to degradation. Intracellular single-chain TSHRs are sensitive to both Endo H and Endo F deglycosylation. [Figure was originally published in K. Tanaka et al: Subunit structure of thyrotropin receptors expressed on the cell surface. J Biol Chem. 1999;274:33979–33984 (27), with permission. © The American Society for Biochemistry and Molecular Biology.]
Figure 4.
Figure 4.
Flow cytometric subtractive assay to estimate the proportion of cleaved vs uncleaved TSHR on the cell surface. Monoclonal antibody 1 to the extracellular component of the B-subunit recognizes both cleaved and uncleaved receptors, as well as in cleaved receptors that have already shed their A-subunits. Monoclonal antibody 2 is to the C-peptide loop present only in the single-chain, uncleaved TSHR. Because this antibody cannot “see” the cleaved, two-subunit TSHR that has lost its C-peptide region, the difference between the signals for antibodies 1 and 2 estimates the proportion of cleaved vs uncleaved TSHR. Potential limitations to interpreting data obtained with this assay are described in the text.
Figure 5.
Figure 5.
Evidence in a mouse model that TSHR A-subunit shedding is required to induce Graves' hyperthyroidism. BALB/c mice were immunized with adenovirus expressing the isolated human TSHR A-subunit or a human TSHR that is unable to cleave into A- and B-subunits (D1-NET; Figure 2A). Control mice (Con) received adenovirus coding for a nonspecific antigen. Mice were injected three times at three weekly intervals. Serum T4 levels were measured at the time of euthanasia 4 weeks after the final adenovirus injection. The shaded area represents the normal range in the control group of mice. Relative to the control mice, the thyroid histology of a representative mouse immunized with A-subunit adenovirus revealed a more columnar and vacuolated epithelium consistent with hyperthyroidism.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pastan I, Roth J, Macchia V. Binding of hormone to tissue: the first step in polypeptide hormone action. Proc Natl Acad Sci USA. 1966;56:1802–1809. - PMC - PubMed
    1. Amir SM, Carraway TF, Jr, Kohn LD, Winand RJ. The binding of thyrotropin to isolated bovine thyroid plasma membranes. J Biol Chem. 1973;248:4092–4100. - PubMed
    1. Buckland PR, Rickards CR, Howells RD, Davies Jones E, Rees Smith B. Photo-affinity labelling of the thyrotropin receptor. FEBS Lett. 1982;145:245–249.
    1. Buckland PR, Howells RD, Rickards CR, Rees Smith B. Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand. Biochem J. 1985;225:753–760. - PMC - PubMed
    1. Kajita Y, Rickards CR, Buckland PR, Howells RD, Rees Smith B. Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane. Biochem J. 1985;227:413–420. - PMC - PubMed

Publication types