Evidence that Cleavage of the Thyrotropin Receptor Involves a "Molecular Ruler" Mechanism: Deletion of Amino Acid Residues 305-320 Causes a Spatial Shift in Cleavage Site 1 Independent of Amino Acid Motif

Tanaka, Kenji

doi:10.1210/en.141.10.3573

Cited by 10 publications

(8 citation statements)

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“…An example is the cleavage of the thyrotropin receptor into two subunits by what is presumed to be a matrix metalloprotease. Cleavage of the receptor by the enzyme occurs at the fixed distance from the protease attachment site (25). Deletion of residues on the receptor at the protease attachment site decreased the cleavage activity and shifted the cleavage site to an upstream region.…”

Section: Discussionmentioning

confidence: 99%

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

Narula

Tse‐Dinh

2012

Nucleic Acids Research

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Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.

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Section: Discussionmentioning

confidence: 99%

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

Narula

Tse‐Dinh

2012

Nucleic Acids Research

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“…Further evidence against cleavase specificity is that the deletion of the TSHR segment (residues 305-320) containing the initial, proximal cleavage site does not abolish cleavage, but shifts the cleavage site further upstream (52). This evidence supports the likelihood that the TSHR cleavase clips the receptor at a fixed distance from the plasma membrane, regardless of the amino acids at the cleavage site.…”

Section: Lack Of or Limited Tissue And Enzymatic Specificity For Tsmentioning

confidence: 89%

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

Rapoport

McLachlan

2016

Endocrine Reviews

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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.

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“…Nevertheless, involvement of such an enzyme remains a distinct possibility. Evidence for a ‘molecular ruler’ mechanism [30] in TSHR cleavage [31] supports a role for a membrane‐associated proteolytic enzyme.…”

Section: Discussionmentioning

confidence: 99%

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor

Chazenbalk

McLachlan

Chen

et al. 2001

European Journal of Biochemistry

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To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125 I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage (< 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317±367) was replaced with the corresponding domain of the TSHR (residues 363±418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317±319 replaced with TSHR-GQE367±369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317±319AAA).In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) aminoacid specificity in a region important for TSHR cleavage (GQE367±369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.

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Evidence that Cleavage of the Thyrotropin Receptor Involves a "Molecular Ruler" Mechanism: Deletion of Amino Acid Residues 305-320 Causes a Spatial Shift in Cleavage Site 1 Independent of Amino Acid Motif

Cited by 10 publications

References 0 publications

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

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

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor

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