A Structure-Function Analysis Revealed Histidine in the Transmembrane Domain of Human Thrombopoietin Receptor Is Essential for a Novel Non-Peptidyl Thromobopoietin Mimetics, NIP-004, To Induce Signaling for Cellular Proliferation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3149-3149
Author(s):  
Akiko Yamane ◽  
Atsushi Miyamura ◽  
Takanori Nakamura ◽  
Norihisa Ishiwata ◽  
Katsuaki Miyaji ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Lines ◽  
Transmembrane Domain ◽  
Function Analysis ◽  
Species Specificity ◽  
Synthetic Compound ◽  
Thrombopoietin Receptor ◽  
Gene Assay ◽  
Agonistic Activity

Abstract NIP-004 is a novel synthetic compound that displays human (Hu) thrombopoietin receptor (Mpl) agonistic activity. NIP-004 stimulated proliferation of HuMpl-expressing cell lines such as UT-7/EPO-HuMpl and Ba/F3-HuMpl, but not murine (Mu) Mpl-expressing cell lines such as UT-7/EPO-MuMpl and Ba/F3-MuMpl. NIP-004 or its derivative compound stimulated colony formation of CD41a+ megakaryocytes from human bone marrow (BM)-derived CD34+ hematopoietic progenitor cells, but not from murine BM cells or cynomolgus or rhesus monkey BM-derived CD34+ cells. These results indicated that NIP-004 has strict species specificity. To identify a molecular basis for the species specificity displayed by NIP-004, we analyzed the amino acid sequence of Mpl from two non-human primates, cynomolgus and rhesus. Cynomolgus Mpl displayed the highest level of sequence homology, 96% identical to HuMpl, with only 22 amino-acids differing between the two species, and 14 residues in the 22 amino-acids of HuMpl differed from rhesus Mpl. Comparison of these 14 amino-acid residues as either hydrophobic or hydrophilic and either electrically charged or uncharged revealed distinguishing features of an amino acid residue. Histidine (His) at position 499 from the N-terminal residue in the transmembrane domain of HuMpl is specific for humans. We thus performed site-directed mutagenesis this residue in HuMpl and MuMpl, and analyzed STAT5 activation via each receptor in the Hek293 human embryonic kidney cell line using STAT-reporter gene assay. Wild-type HuMpl, but not MuMpl, activated STAT5 after stimulation with NIP-004. HuMpl with His499 mutated to Leu (HuMplH499L) failed to induce STAT5 activation via NIP-004 stimulation. Conversely, when MuMpl was engineered to contain His490 (MuMplL490H), it was then capable of activating STAT5. Furthermore, NIP-004 stimulated proliferation of Ba/F3-MuMplL490H cells, but not Ba/F3-HuMplH499L cells. Western blotting analysis revealed that NIP-004 induced phosphorylation of STAT5 proteins in Ba/F3-HuMpl and Ba/F3-MuMplL490H cells, but not Ba/F3-MuMpl and Ba/F3-HuMplH499L cells. All Ba/F3 transfectants expressing these Mpl constructs responded to TPO stimulation. These observations indicate that His in the transmembrane domain of Mpl is an essential residue for the ability of NIP-004 to act as an Mpl agonist. We also found none of other experimental animals such as common marmoset, squirrel monkey, beagle dog, Japanese White rabbit, Syrian hamster, Hartley guinea pig and Wistar rat with His in the transmembrane of Mpl. Although His exists in the transmembrane domain of human G-CSFR, NIP-004 failed to stimulate the proliferation of Ba/F3 cells expressing human G-CSFR. In conclusion, a novel non-peptidyl synthetic compound, NIP-004, displays Mpl agonistic activity with strict species specificity via His in the transmembrane domain of Mpl.

Sequence of the rat factor VIII cDNA

2004 ◽  
Vol 91 (01) ◽  
pp. 38-42 ◽  
Author(s):  
Christof Geisen ◽  
Erhard Seifried ◽  
Johannes Oldenburg ◽  
Matthias Watzka
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Factor Viii ◽  
Posttranslational Modifications ◽  
Function Analysis ◽  
Amino Acid Level ◽  
Coagulation System ◽  
Cleavage Sites ◽  
Human Factor Viii ◽  
Exon 20

SummaryFactorVIII acts as an essential compound of the tenase complex of the coagulation system. Herein we report the cDNA of the rat factor VIII. The rat cDNA comprises 6777 nucleotides and encodes a protein of 2258 amino acids, 61 amino acids less than mouse and 92 amino acids less than human factor VIII. The overall identity compared to human cDNA is 61% on the cDNA and 51% on the amino acid level. In cDNA, highest levels of sequence identity can be observed in the A and C domains (ranging between 68% and 73%), whereas B domain and the small acidic regions are more divergent (34%-49%). Compared to mouse and human most sites for posttranslational modifications such as sulfatation and glycosylation as well as thrombin and protein C cleavage sites are conserved in rat. Alternative transcripts lacking exon 17 and/or comprising additional 26 bp due to alternative splicing of exon 20 were found. Furthermore, 13 polymorphisms (seven in exon 14, one in exon 20, 23, 24, and 25, two in the 3’UTR) three of which lead to an amino acid exchange could be detected. Our findings might provide new insights into the structure-function analysis of the factor VIII protein and might prove useful for future animal models addressing the function of factor VIII.

scholarly journals Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2634-2643 ◽  
Author(s):  
Vahid Afshar-Kharghan ◽  
José A. López
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Silent Mutation ◽  
Cell Surface Expression ◽  
Unaffected Individual ◽  
Exon 2 ◽  
The Third ◽  
Bernard Soulier Syndrome

We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.

scholarly journals Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor

2002 ◽  
Vol 76 (16) ◽  
pp. 7976-7986 ◽  
Author(s):  
Valerie M. Nappi ◽  
Lisa M. Petti
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Growth Factor ◽  
Transmembrane Domain ◽  
Specific Interaction ◽  
Platelet Derived Growth Factor ◽  
Helical Conformation ◽  
Bovine Papillomavirus ◽  
E5 Protein ◽  
Amino Acid Requirements

ABSTRACT The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor β receptor (PDGFβR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFβR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFβR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFβR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFβR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.

The Effect of Cell Population Density on Nutrient Uptake and Cell Metabolism: a Comparative Study of Human Diploid and Heteroploid Cell Lines

1972 ◽  
Vol 10 (2) ◽  
pp. 515-524
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Dna Synthesis ◽  
Nutrient Uptake ◽  
Cell Lines ◽  
Acid Concentration ◽  
Contact Inhibition ◽  
Inhibition Of Growth ◽  
Extracellular Amino Acid

The possibility that contact inhibition of growth in cultures of human diploid cells is influenced by the effects of cell crowding on nutrient uptake by the cells was investigated. Two human lung cell lines were compared, the diploid line MRC-5 and the heteroploid line L-132. In pre-confluent cultures the ability of these 2 cell types to accumulate amino acids was very similar. Post-confluent L-132 cells showed very little change from the pre-confluent cultures but the ability of MRC-5 cells in post-confluent cultures was greatly reduced. The intracellular concentrations of various amino acids necessary to achieve the maximum rate of protein synthesis were found. These values were identical for sparse and crowded cultures but due to the reduced uptake ability of crowded MRC-5 cells a far higher external amino acid concentration was required in post-confluent cultures. This meant that although amino acids did not become growth-limiting until over 80% utilized in pre-confluent cultures, in post-confluent cultures they became growth-limiting when only 50% utilized. Although protein synthesis was significantly affected by extracellular amino acid concentration and cell crowding, thus contributing towards the effect of contact inhibition of growth, DNA synthesis was shown to be the major metabolic function in contact inhibition. Increased cell density had a very inhibitory effect on DNA synthesis in MRC-5 cultures, but not in L-132 cultures, and this was unaffected by extracellular amino acid and glucose concentration.

scholarly journals Prognostic Significance of Serum Free Amino Acids in Head and Neck Cancers

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 428 ◽  
Author(s):  
Vit Vsiansky ◽  
Marketa Svobodova ◽  
Jaromir Gumulec ◽  
Natalia Cernei ◽  
Dagmar Sterbova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Regression Model ◽  
Head And Neck ◽  
Cell Lines ◽  
Proportional Hazards ◽  
Cox Proportional Hazards ◽  
Transformed Cells ◽  
Cox Proportional Hazards Regression ◽  
Proportional Hazards Regression

Despite distinctive advances in the field of head and neck squamous cell cancer (HNSCC) biomarker discovery, the spectrum of clinically useful prognostic serum biomarkers is limited. As metabolic activities in highly proliferative transformed cells are fundamentally different from those in non-transformed cells, specific shifts in concentration of different metabolites may serve as diagnostic or prognostic markers. Blood amino acids have been identified as promising biomarkers in different cancers before, but little is known about this field in HNSCC. Blood amino acid profiles of 140 HNSCC patients were examined using high-performance liquid chromatography. Cox proportional hazards regression model was used to assess the prognostic value of amino acid concentrations in serum. Colony forming assay was used to identify the effect of amino acids that were significant in Cox proportional hazards regression models on colony forming ability of FaDu and Detroit 562 cell lines. In the multivariable Cox regression model for overall survival (OS), palliative treatment was associated with an unfavourable prognosis while high serum levels of methionine have had a positive prognostic impact. In the relapse-free survival (RFS) multivariable model, methionine was similarly identified as a positive prognostic factor, along with tumor localization in the oropharynx. Oral cavity localization and primary radio(chemo)therapy treatment strategy have been linked to poorer RFS. 1mM serine was shown to support the forming of colonies in both tested HNSCC cell lines. Effect of methionine was exactly the opposite.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

A Novel, Small Non-Peptidyl Butyzamide Activates Human Thrombopoietin Receptor and Promotes Megakaryopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2201-2201 ◽  
Author(s):  
Wataru Nogami ◽  
Hiroshi Yoshida ◽  
Kenzo Koizumi ◽  
Hajime Yamada ◽  
Kenji Abe ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Fetal Liver ◽  
Human Platelets ◽  
Stable Transfectants ◽  
Human Cd34 ◽  
Thrombopoietin Receptor ◽  
Cd34 Cells ◽  
Agonistic Activity ◽  
Nog Mice ◽  
Orally Bioavailable

Abstract Butyzamide is a novel non-peptidyl molecule which has agonistic activity to the thrombopoietin (TPO) receptor Mpl. Butyzamide promotes the proliferation of murine pro B cell line Ba/F3 expressing human Mpl (hMpl), and induces phosphorylation of JAK2, STAT3, STAT5 and MAPK. Interestingly, butyzamide does not promote the proliferation of Ba/F3 cells expressing murine Mpl (mMpl). To elucidate the mechanisms for this species specificity, we created stable transfectants such as Ba/F3-hMpl(H499L) and Ba/F3-mMpl(L490H) cells. Butyzamide induced the proliferation of Ba/F3-mMpl(L490H) but not Ba/F3-hMpl(H499L) cells, indicating that a histidine residue in the transmembrane domain of human Mpl is critical for butyzamide-induced signaling. Butyzamide induced the colony-forming unit-megakaryocyte and polyploid megakayocytes from human CD34+ hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was orally administered to immunodeficient NOD/Shi-scid/IL-2Rγcnull (NOG) mice transplanted with human fetal liver-derived CD34+ cells, the human platelet count increased by 6.2- and 22.9-fold at the doses of 10 and 50 mg/kg for 20 days, respectively. Butyzamide also increased the number of reticulated human platelets and human matured megakaryocytes in NOG mice. These results indicate that butyzamide is an orally bioavailable Mpl activator and suggest its potential for clinical development as a therapeutic agent in patients with thrombocytopenia.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

scholarly journals Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function

2003 ◽  
Vol 369 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Xiang Y. LIU ◽  
Teah L. WITT ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Transmembrane Domain ◽  
K562 Cells ◽  
Reduced Folate Carrier ◽  
Wild Type ◽  
Linker Region ◽  
Conserved Amino Acids

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1—6 and TMDs 7—12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204—214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFCD215—R263Δ, hRFCK204—R263Δ and hRFCK204—R214Δ proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6S)5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFCK204—R263Δ restored low levels of transport (16—21% of the wild type). Insertion of the ThTr1 linkers into hRFCD215—R263Δ at position 215 restored 60—80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts

1997 ◽  
Vol 18 (2) ◽  
pp. 101-112 ◽  
Author(s):  
D W Silversides ◽  
A Houde ◽  
J-F Ethier ◽  
J G Lussier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Full Length ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pcr Cloning ◽  
Specific Expression ◽  
Terminal Domain ◽  
Bovine Tsh

ABSTRACT The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86·4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane α-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n=22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9·3 and 4·3 kb, and a minor transcript of 3·8 kb being detected.

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