GB2376016A

GB2376016A - Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10

Info

Publication number: GB2376016A
Application number: GB0113030A
Authority: GB
Inventors: Nadir R Farid; Viktoria Kaczur; Lazlo G Puskas
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-05-30
Filing date: 2001-05-30
Publication date: 2002-12-04
Also published as: GB0113030D0

Abstract

Thyrotropin receptor (TSHR) is enzymatically cleaved at residues 367-396 by the metalloprotease ADAM 10. A 16 amino acid peptide encompassing the ADAM 10 is identified, which blocks ADAM 10 cleavage of the receptor in an assay. Variants and mutants of such a peptide are claimed. Also identified is a ferritin binding region in TSHR. Alternatively claimed are nucleic acid probes that can be used in assays, such as on an array, to identify different metalloproteases of, for example the ADAM and ADAMTS families.

Description

THE THYROTROPIN (TSH) RECEPTOR CLEAVING ENZYME, ITS RECOGNITION MOTIF AND DOMAINS WITHIN IT CONTRIBUTING TO RECEPTOR SIGNALLING

BACKGROUND The TSH receptor is a member of a rapidly expanding family of leucine rich repeat containing G protein-coupled (LGR) receptors. It is most closely related to the luteneizing hormone (LHR) and follicle-stimulating hormone (FSHR) receptors whose ligands share structural similarities to TSH. In contrast to FSH and LH receptors, the TSH receptor has an 50 aminoacid"add on"sequences in the Cflanking region of its extra-cellular domain (ectodomain). The TSHR ectodomain is enzymatically cleaved. The-flanking region"add on"sequences (CFLANS), although not necessary for TSH binding or signalling appears to be necessary for cleavage (1). The evidence to date does not support the notion that TSHR cleavage is physiologically regulated and there is no clear understanding as to the consequences of TSHR cleavage. There are currently two demonstrated cleavage sites-one N-terminal to the CFLANS region and the second just outside its Cterminal. There is no compelling evidence as to which of the two sites is the primary digestion site and whether either of the two cleavage sites is actually regulated (see 1 for review) Apparently after cleavage, disulphide bonds still hold the two TSHR subunits, (2). A surface protein disulphide isomerase (PDI) is then involved in the disulphide bond reduction. Depending on the redox state of the cellular micro-environment PDI functions as a chaperone as well as an facilitator of re-oxidization of reduced disulphide bonds (3,4).

Based on earlier findings (5), we suggest that following cleavage PDI is involved in forming disulphide bonds between adjacent B subunits, to enhance signalling of thyroid cells. This mechanism may well compensate for the surprisingly (-10, 000) of TSHRs on thyroid cells.

We thus suggest a sequential series of events in TSH binding to and signalling through the receptor: 1) Binding of TSH to the ectodomain 2) Cleavage of TSHR to

terminate signal initiated by ectodomain conformational changes 3) Reduction of disulphide bonds between cleaved subunits 4) Covalent dimer formation between B subunits to magnify signalling 5) Termination of hormone specific signalling by way of desensitisation and receptor internalization.

The TSHR cleavage enzyme has yet to be identified (1,6, 7). Building on previous studies, we postulated that TSHR cleavage enzyme is likely to be a member of the ADAM/ADAMTS family. To that end, we constructed 56-60 mers specific for 27 family member enzymes (Figure 1), for which sequences were deposited in Human Genome database. These enzyme-specific oligonucleotides are the subject of a patent application. The oligonucleotides were used to probe the expression of their cognate enzymes in a pool of normal thyroid tissue RNA, as well as in placenta and fetal brain RNA as controls (figure 2 a, b, c). A number of enzymes, including ADAMS 10 and ADAMS TS1 (which were noted to be expressed in thyroid tissue) were expressed in thyroid. These two enzymes were known to be expressed in thyroid tissue (http :// www. ncbi. nih. gov). ADAM 10 was not expressed in the other two control tissues, whereas ADAM 9 and 17 which have been incriminated in the cleavage (or shedding) of many other receptors.

We thus further focused on ADAM10, its recognition site on TSHR and its role in TSHR cleavage. First, we searched for homology within the CFLANS region of TSHR and other surface receptors or proteins known to be cleaved by ADAM10 (8).

A conserved motif was identified over TSHR 337-355 region (Figure 3). We custom-synthesised a 16 residue peptide corresponding to this motif and, as a control, a scrambled peptide (Figure 4). The peptide corresponding to the ADAM10 binding motif but not the scrambled control inhibits in a dose-dependant manner receptor cleavage at residues 367-369.

Apparently the ADAM enzymes require highly reactive oxygen for their activation (9), likewise PDI figures importantly in the sequence of events leading to TSHR signalling. We searched for and found a domain in CFLANS which show similarities

to the H chain of ferritin (Figure 5). This domain may be important in the generation and maintenance of redox states important for TSHR processing and thus function.

MATERIALS AND METHODS

C o n s t r u c t Z o n o f M Z c r o a r r a y s. O l Z g o n u c I e o t Z d e Construction ofmicroarrays. Oligonucleotide sample design and synthesis.

Human control clones were obtained from a mixed human library, cloned in pBluescript SK II (-) plasmid (New England Biolabs) with traditional techniques (10) and both strands were sequenced by dye-terminator method and vector-specific primers (T7 and T3 primers) on an automated sequencer (ABI, Applied Biosystems) Human control cDNA inserts were amplified with M13 forward and reverse primers in 2x 100 III of total volume, amplicons were analysed on a 1.5% agarose gel, ethanol-precipitated, washed two times with 70% ethanol, dried and resuspended in 50% DMSO/water at a final concentration of 0.8 mg/ml.

For ADAM-and ADAMTS-specific samples 56-60 nucleotide long synthetic oligonucleotides were prepared. Oligonucleotides with homopolymeric stretches of more than six bases, oligonucleotides with low Tm-points or with strong secondary structure were rejected. To avoid self-complementary sequences OLIGO 4.1 primer analysis software (National Biosciences Inc. ) was used. All the sequences were checked in the sequence database (http :// www. ncbi. n1m. 1Úh. gov) with the BLASTN program for complementarity to other human sequences (11). All the sequences were excluded which contained minimum 16 nt long complementary region to any human cDNA sequences or human introns including repetitive elements. Oligonucleotides were synthesized using standard phosphoramidite methodology on an Expedite Synthesizer (ABI, Applied Biosystems). The oligonucleotides were purified on OligoPack columns (Glen Research) and dissolved in 50% DMSO/water at a final concentration of 0.9 mg/ml or 0.1 mg/ml. The quality of theoligonucleotides were checked with HPLC and/or mass spectrometry. The sequences of the oligonucleotid samples can be seen in Figure 1 and their accession numbers along of those of control genes in Table 1. Amplified cDNA inserts and synthetic oligonucleotides were arrayed on amino-silanized slides (Sigma) by using a MicroGrid Total Array System (BioRobotics, UK) spotter. 26 PCR amplified cDNA clones, lambda phage

DNA (0.8 mg/ml in 50% DMSO) (Fermentas), T4 dc DNA (Takara) (0.6 mg/ml in 50% DMSO), two Cy5-labelled 34-mer oligomer (labelled at its 5'-end, 10 pmol/ l) and 27 ADAM-and ADAMTS-specific oligomers were spotted in duplicates onto slides.

GENERATION OF MICROARRAY PROBES, SAMPLE AMPLIFICATION.

Total RNAs were purified by SV Total RNA isolation kit (Promega) according to the manufacturer's instructions. 200 u. g of total RNA from placenta and 450 u. g of total RNA from thyroid glands were obtained. The purity of the RNA was confirmed by agarose-gelelectrophoresis RNA amplification method according to Barry and Brown (12). In first strand cDNA synthesis, 8 ig of total human placenta RNA, or 8 u. g total human fetal brain RNA (Clontech) or 8 g total human thyroid RNA was mixed with 2 g of an anchored oligo-dT primer (HPLC-purified) having T7 promoter sequence at its 5'-end, 40 units ofRNasin (Fermentas) in a total volume of 14 u. I, heated to 750C for 5 min and cooled on ice To this mixture, 4 l 5x first

strand buffer (Promega), 1 u. I 10 mM dNTP mix and 1 u. I, 200 Units of RnaseH (-) point mutant M-MLV reverse transcriptase (Promega) was added in 20 pI total volume. Samples were incubated at 42 C for 2 hours. To the first strand reaction mix 103. 8 u. I water, 33. 4 11 5x second strand synthesis buffer (Gibco), 3. 4 11 10 mM dNTP mix (Amersham Pharmacia), 1 u. I E. colz DNA ligase (Gibco, 10 U/u.l), 4 u.l E. coli DNA Polymerase I (Gibco, 10 U/il) and I ul E. colz RNAse H (Gibco, 2 U/) 11) was added, and incubated at 16 C for 4 hours. The synthesized double-stranded DNA was purified with PCR purification kit (Qiagen). After sample concentration by vacuum, antisense RNA synthesis was done by RiboMAX in vitro transcription kit (Promega) in total volume of 30 l according to the manual. The RNA was purified with NucleoSpin RNA purification kit (Macherey-Nagel), concentrated and measured spectroscopically. 4 u. g of amplified RNA was used in first-strand synthesis for generation of cDNA probes. Cy5-dTTP (Amersham Pharmacia) (0.05

mM in reaction mix) were incorporated during reverse transcription using random heptamers (2 u. g), 0. 3 mM d (G/C/A) TPs, 0. 06 mM dTTP, Ix first strand buffer, and 200 Units of RnaseH (-) point mutant M-MLV reverse transcriptase (Promega) in 20 l total volume. The RNA and random primers were preheated for 75 C for 5 min and cooled in ice before adding the remaining reaction components. After 2 hours incubation at 37 C, the heteroduplexes were denaturated and the mRNA was hydrolysed with NaOH (25 mM final concentration, 15 min. at 37 C). After

neutralization of the mixture with 10 41 of 2 M MOPS buffer (pH 5. 0), the labelled cDNA was purified with Nucleospin PCR purification kit (Macherey-Nagel) according to the manufacturer's instructions, except that the probes were eluted by two times addition of 50 pLI of deionized water. The labelled probes were recovered by vacuum concentration, and redissolved in 20 ! of Huntsman Hybridization solution (50 % formamide, 5x SSC, 0. 1 % SDS, 100 g/m ! salmon sperm DNA).

ARRAY HYBRIDIZATION AND POSTHYBRIDIZATION PROCESSES.

For hybridization, spotted slides were denatured in boiling water for 2 min, dehydrated in ethanol for 10 sec, dried, UV cross-linked and blocked with 2% BSA in Ix SSC, 0.2% SDS solution at 42 C for 30 min, then washed with water.

Prehybridization was done by adding 1 pLI of 4 mg/ml poly (T), 2 il of 2 mg/ml human Cot DNA (Gibco) and 2 ptl of I mg/ml Lambda DNA (Fermentas) to the probe mix, and incubated at 42 C for 30 min after denaturation by heating for 5 min at 80 C. 20 cl of the mix was placed on the blocked array under a 24 mm x 32 mm coverslip. To the sides of the coverslip DPX Mountant (Fluka) was poured in order to prevent evaporation. Slides were incubated at 42 C for 18 hours in a humid hybridization box. The mountant was removed and the arrays were washed by submersion and agitation for 10 min in Ix SSC with 0. 1 % SDS, for 10 min in 0. 1x SSC with 0.1% SDS and for 10 min in 0.05x SSC at room temperature, then rinsed briefly in deionised water and dried with high pressure nitrogen gas.

SCANNING AND DATA ANALYSIS.

Each array was scanned under a green laser (532 nm) (in the case of Cy5 labelling) by using a ScanArray Lite (GSI Lumonics) scanning confocal fluorescent microscope with 10 gm resolution. Image analysis was performed by ScanAlyze2 software (11). Each spot was defined by manual positioning of a grid of circles over the image. The average pixel intensity and the local background of each spot were determined. Data analysis was done by Microsoft Excel software (Microsoft). For background corrections those data were calculated as negatives where the average intensity of the spot minus three times standard deviation was smaller than five times of the average background of the same area. Those results were also excluded where the replicate spots from different site of the same array or results from replicate experiments were significantly different. Spots having five times higher than the average background (calculated from local backgrounds) were visualized in by ScanAnalyze and Jasc Paint Shop Pro Software 7.00 (Jasc Software Inc. ) (Figure 2 a-c)

TSHR CLEAVAGE CHO cells transfected with human TSHR (CHO-WT) (13) were grown in DM medium supplemented with 10% fetal calf serum (FSC)-and antibiotics Cells were removed mechanically from culture dishes and collected directly into ice-cold buffer containing 250 mM sucrose, 1.25 mM EGTA, 50 mM, pH7.6 containing tablets of freshly dissolved proteinase inhibitor cocktail tablets according to the manufacturer's (Roche,..., Switzerland) Buffer A]. The cells were homogenized on ice and centrifuged at 760 g for 10 min. The plasma membranes fraction was pelleted by ultracentrifugation at 250,000g for 1 hour. lOOg plasma membrane in buffer A containing 2.5 mM ZnC12 were incubated for 2 hours at 37 C with gentle shaking in the absence or presence of TSH with or without the peptides shown in figure 4.

The samples were ran on SDS-PAGE in the prescience of reductant using 12.5% on Pharmacia-Hoeffer Tall Mighty Small. The protein bands thus resolved were transferred to PVDF membranes for 1.4 hours at 100 V/200mA. The non-specific

sites were blocked with 5% non-fat dried milk 0. 1% (V/V) Tween 20 in PBS in 1hour at 37 C on an orbital shaker. After rinsing twice with wash buffer, the membranes were incubated with 5 mls of PBS 10% FCS, 10% glycerol, 1 moll glucose and 0.5% Tween 20 containing 1Jlg/ml of either RSR-1 or RSR-2 monoclonal antibody (RSR Ltd, Cardiff, UK) (14) and developed using antimouse IgG horseradish peroxidase conjugate followed by enhanced chemiluminescence reagents. RSR-2 was directed at an epitope encompassing 246-260 of hTSHR and RSR-1 at residues 381-385.

TABLE 1 - GENES STUDIED ADAM/ATAMS FAMILY ACCESSION NUMBER NAME ADAM2 NM 001464 ADAM3A AJ005372 ADAM5 AJ AJ132825 ADAM6 AC007028 ADAM8 D26579 ADAM9 D14665 ADAMI 0 AF009615 ADAM11 AB009675 ADAM12 AF023476 ADAM17 U69611 ADAM18 XM 011685 ADAM19AF134707 ADAM20 AF158643 ADAM21AF 158644 ADAM22 XM 004595 ADAM23 AF158641 ADAM28 AF137334 ADAM29 AF171929 ADAMTS2 XM 003934 ADAMTS3 AB002364 ADAMTS4 AB014588 ADAMTS5 AF141293 ADAMTS6 AF140674 ADAMTS7 AF140675 ADAMTS8 NM 007037 ADAMTS9 AF261918 CONTROL GENES Translation initiation factor 4A (eIF-4A) isoform 2 D30655 Human mRNA for KIAA0162 gene U46691 Homo sapiens mRNA for cystinosin AJ222967 Thyroid peroxidase (alternative products) J02969 Deoxyribonuclearse I precursor M55983 DNA topoisomerase I U43431 CD58 antigen (lumphocyte function associated antigen 3) Y00636 Human mRNA for KIAAA0025 gene AF055001 Sodium channel, voltage-gated, type VI, alpha polypeptide M91556 Bone morpho-genetic protein receptor, type IB U89326 Thyroid receptor interactor (TRIP3) L40410

Nuclear pore complex protein nhup153 Z25535 Sodium channel protein, brain specific NM 002976 ThymopoietinU18271 Dihydrofolate reductaseJ00140 Homeobox protein Cdx2 U51096 Ryanodine receptor 2 X98330 Lambda Phage DNA NC~001416 Angiotensin II receptor S77410 Cyclin E M74093 Myosin binding protein-C (MyBP-C) U91629 IPW MRNA sequence U12897 Protein phosphatase 2A beta subunit M64930 Glycerol kinase 2 (testis specific) X78712 T4 dc DNA NC 000866 Apoptotic cysteine protease Mch4U60519 Acyl-coA dehydrogenase AF037438 Ric (Drosophilia)-like (expressed in neurons) U78166

REFERENCES rapport B, Chalzenbalk, G. D. , Jaume J C, McLachlan, S M 1998 Endocrine Reviews 19: 673 2 Couet, J De Bernard S Loosfelt H, Saunier B, Milgrom E, Mishari M. 1996 Biochemistry 35 14800 3 Frand, A. R. , Cuozzo, J. W., Kaiser C.. 2000 Trends in Cell Biology 10 203 4 Tsai B, Rodigheiro Co. Lencer W. I, Raporort T. a. 2001 Cell 104: 937

5 Islam N. M., Farid, N. R. 1985Experientia4118 6 De Bernard S, Mishari M., Huet J-C., Beau I., Desroches A, Loosfelt H, Pichon C, Pemollet J-C., Milgrom E. 19991. Bioi Chern. 274'101 7 Tanaka K, Chazembalk G. D, McLachlan S. M., Rapoport B 1999 Mol Cell Endocrinology 150: 113.

8 Black, R. A. , White J. M. 1998 Current Opin Cell Biolosy 10 : 654.

9 Zhang, Z. , Oliver P. , Lancaster 1. R. Jr. , Schwartzenberger P. O. , Joshi M. S. , Corks J., Kolls J. K.

2001 FASEB J. 15.303.

10 Sambrook J, Fritsch, E. F. , Maniatis T. Molecular cloning : a laboratory manual Cold Spring Harbor Press, Cold spring Harbor, NY, 1998 11. http :// www. microarrays. org/software. html

12 Barry C, Brown PO 1999 Modified Eberwine (Antisense") RNA amplification protocol. http//www microarrays. org/pdfs/ModifiedEberwine. pdf 13. Shi Y, Zou M, Parhar RS, Farid NR 1993 Thyroid 3 129. 14. Oda Y, sanders J, EvansM, Kiddie A, Munkley A, James C, Richards T, Wills J, Furmaniak J, Smith BR 2000 Thyroid 10: 1051.

Claims

THE CLAIM 1. That ADAM 10 is the long-sought after TSHR cleaving enzyme.

We claim priority to identifying the TSHR cleaving enzyme and the definitive site of cleavage at 367-369, any modification for experimental or therapeutic purposes which would modify (enhance or cripple), the enzyme activity in thyroid tissue or modifications of the TSHR site by whatever means which would modify its cleavage.
2. The 16 AA peptide corresponding to ADAM 10 recognition motif.

Homologues whether proteineous or resulting from translation of DNA or RNA precursor the use of such peptides or derivatives either in experimental or therapeutic settings to modify the cleavage of TSHR or related structures.

We further lay claim to the introduction of such a motif by whatever means into proteins which do not contain for the purpose of creating a novel cleaving site for ADAM 10. This claim extends to the artificial introduction of this site into other structures for the purpose of initiating cleavage.
3. The enzyme-specific probes (56-60 mers) used to identify: ADAMS 2, 3 A, 5,6, 8,9, 10,11, 12,17, 19,20, 21,22, 23,28, 29 and ADAM TS 1,2, 3,4, 5,6, 7,8, and 9.
4. Likewise we claim sequences based on the ferritin-like domain and any modification thereof in TSHR or related structures, its introduction into other structures or its use as peptide to modify the activity of those receptors or related structure.