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Biomedical subjects

P R Buckland

Publications and source records attributed to P R Buckland.

At least 37 records · Page 2Linked to original sources
PubMed1991

The effects of antidepressant drugs on kainate receptor mRNA levels.

We have measured the levels of kainate receptor mRNA in rat brains following the injection of antidepressant drugs over a time course up to 32 days. Over this period imipramine and fluvoxamine elicited a rise in kainate receptor mRNA levels to a maximum of three and two fold increase respectively, relative to untreated rat brain samples. Mianserin and amitriptyline did not induce significantly elevated kainate mRNA levels compared to untreated specimens, but were elevated compared to vehicle injected controls.

Animals↗
PubMed1988

Isolation and characterization of the gene encoding the alpha-subunit of the rat pituitary glycoprotein hormones.

The gene encoding the common alpha subunit of the rat pituitary glycoprotein hormones was isolated from a rat genomic DNA library. The gene spans approximately 8 kb, and contains four exons and three intervening sequences of 5.4 kb, 1.1 kb and 0.6 kb. Blot hybridization of restriction enzyme digests of rat genomic DNA suggests that the alpha gene is present in a single copy. The coding region and 424 bp of the 5'-flanking region of the gene were sequenced. Primer extension and S1 nuclease analyses revealed a single transcriptional start point downstream from consensus promoter elements. The organization of the rat alpha-subunit gene is similar to that of the human and bovine genes including the sizes and locations of the four exons and three introns. In addition, a region of strong sequence similarity has been identified in the 5'-flanking region of the rat, human and bovine genes. This region includes sequences which are similar to a putative triiodothyronine regulatory element and the previously identified cAMP regulatory region; such sequences may mediate the known effects of these factors on alpha-subunit gene expression.

Animals↗
PubMed1987

Photoaffinity labelling of the TSH receptor on FRTL5 cells.

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

Affinity Labels↗
PubMed1986

Thyrotropin cross-links to the thyrotropin receptor through both the alpha and beta subunits.

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.

Azides↗
PubMed1986

Assessment of the shape and molecular size of TSH-TSH receptor complexes.

Photoaffinity labelling and analysis under denaturing conditions (SDS-PAGE) have shown that the porcine TSH receptor contains an A subunit (Mr = 47,000) which forms the binding site for TSH and a B subunit (Mr = 25,000) linked to the A subunit by a disulphide bridge. In order to assess the size and shape of the receptor under non-denaturing conditions we have solubilized photoaffinity-labelled porcine TSH receptors using the small micelle-sized detergent sodium deoxycholate and analysed the preparations by sucrose density gradient centrifugation and gel filtration. Under these conditions, the cross-linked TSH-TSH receptor complex showed an S20,w of 6.4 S and a frictional ratio f/f0 of 1.8. These values were consistent with those which might be expected from an elongated protein complex with a molecular weight of about 100,000 (the value obtained by SDS-PAGE). Analysis of another thyroid membrane protein, human thyroid microsomal antigen (Mr = 110,000 by SDS-PAGE) under the same conditions gave an S20,w of 6.0 S and f/f0 = 1.3, suggesting that this protein has a compact structure. The TSH receptor A subunit cross-linked to TSH (Mr = 70,000 by SDS-PAGE) gave an S20,w of 4.6 S and f/f0 = 1.8 and these values could be compared with those obtained for the A subunit alone (S20,w = 3.6 S; f/f0 = 1.4; Mr by SDS-PAGE = 47,000) and TSH alone (S20,w = 2.6 S; f/f0 = 1.6; Mr = 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Characterization of TSH antagonist activity in the serum of patients with thyroid disease.

The ability of sera from patients with thyroid disease to block TSH stimulation of cyclic AMP release from isolated porcine thyroid cells has been assessed and the blocking activity characterized. TSH receptor binding activity was also measured. No blocking or receptor binding activity was detectable in patients with primary myxoedema (n = 23), Hashimoto's disease (n = 11), multinodular goitre (n = 6), or rheumatoid arthritis (n = 10). However, analysis of sera from 23 patients (out of an initial screen of 110 patients) with treated Graves' disease which did not stimulate cyclic AMP production in the bioassay showed that two of these sera contained powerful blocking and receptor binding activity. Both these patients had been treated with 131I. Analysis of the two sera by gel filtration on Sephadex G-200 indicated that blocking and TSH receptor binding activity were associated only with the IgG fraction. Digestion of the IgG with pepsin followed by reduction showed that both (Fab)2 and Fab fragments contained high levels of blocking and binding activity. Antibody divalency was not necessary therefore for TSH antagonist activity. However, our studies suggest that autoantibodies of this type with TSH antagonist activity do not occur frequently in patients from the Cardiff region with primary myxoedema, Hashimoto's or treated Graves' disease.

Biological Assay↗
PubMed1985

Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor.

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

Autoantibodies↗
PubMed1985

Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

Affinity Labels↗
PubMed1985

Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand.

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.

Affinity Labels↗
PubMed1985

Immunoprecipitation of TSH-TSH receptor complexes.

The ability of Graves' sera to interact with the TSH receptor crosslinked to a 125I-labelled photoactive derivative of TSH has been investigated. Crosslinked complexes were prepared using non-purified detergent solubilized human thyroid and guinea pig fat TSH receptors. Affinity purified porcine TSH receptor preparations wee also used. After crosslinking, the crosslinked TSH-TSH receptor complexes were separated from aggregates and free TSH on Sephacryl S-300, incubated with test sera followed by immunoprecipitation using anti-IgG or Protein A. Using non-purified human TSH receptors crosslinked to TSH, a mean +/- SD of 12.1 +/- 4.9% of the crosslinked complex was immunoprecipitated with Graves' sera (n = 7) compared with 10.3 +/- 2.6% with Hashimoto sera (n = 6; P greater than 0.14) and 3.8 +/- 1.0% with normal sera (n = 6; P less than 0.004). These values were markedly reduced when TSH receptor preparations free of other thyroid autoantigens (guinea pig fat TSH receptors) were used. Under these conditions immunoprecipitation with Graves' sera (n = 24) was 1.6 +/- 1.3% compared with 0.8 +/- 0.6% for Hashimoto sera (n = 13) and 0.8 +/- 0.4% for normal sera (n = 12; P less than 0.003). In addition complexes formed between TSH and affinity purified porcine TSH receptors gave low immunoprecipitation values for Graves' (1.44 +/- 0.73%; n = 20) and Hashimoto sera (1.7 +/- 0.94; n = 11) which were not significantly different (P greater than 0.4). Overall, therefore, the effects of Graves' and Hashimoto sera were similar and the amounts of material immunoprecipitated were markedly reduced when TSH receptor preparations containing reduced amounts of other autoantigens were used. Consequently the Graves' sera did not appear to interact specifically with crosslinked TSH-TSH receptor complexes. However the Graves' sera studied did contain TSH receptor antibodies which could inhibit the binding of labelled TSH to TSH receptors in the preparations used and our results suggest that the binding of TSH and these antibodies to the receptor is mutually exclusive. There is considerable evidence that serum from patients with Graves' disease contains antibodies to the TSH receptor (Rees Smith, 1981). Several studies have suggested that binding of the receptor antibody and TSH to the TSH receptor is mutually exclusive (Manley et al., 1977; Petersen et al., 1977; Rickards et al., 1981) but recently the formation of termolecular complexes consisting of detergent solubilized receptors, labelled TSH and Graves' IgG has been reported (Konishi et al., 1982; De Bruin et al., 1984).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

TSH crosslinks to the TSH receptor through the beta subunit.

TSH binds specifically to the TSH receptor present on the surface of thyroid follicular cells and consequently activates adenylate cyclase. It was not known, however, which of the two subunits of the TSH bound to the receptor. By covalently crosslinking TSH to its receptor and characterizing the product with antisera specific for either the alpha or beta subunit of TSH, we have shown that the TSH beta subunit but not the TSH alpha subunit crosslinks to the TSH receptor.

Animals↗
PubMed1985

Thyrotropin receptor antibodies.

The thyrotropin (TSH) receptor is an integral membrane protein which contains 2 subunits linked by a disulphide bridge. The A subunit (mol. wt. 50,000) is water soluble and forms the binding site for TSH, whereas the B subunit (mol. wt. 30,000) penetrates the lipid bilayer and probably forms the site for interaction with adenylate cyclase. Autoantibodies to the TSH receptor are found in the sera of patients with Graves' disease. The antibodies bind to the same region of the receptor's A subunit as TSH and usually act as TSH agonists, causing hyperthyroidism. Occasionally, TSH receptor autoantibodies are found which can act as TSH antagonists. Isoelectric focusing and binding studies indicate that these antibodies also bind to the same region of the receptor A subunit as TSH.

Animals↗
PubMed1984

A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling.

TSH receptors from guinea pig thyroid and epididymal fat have been covalently crosslinked to 125I-labelled TSH conjugated to N-hydroxysuccinimidyl-4-azidobenzoate. Analysis by SDS-PAGE and autoradiography showed bands corresponding to TSH subunits (Mr 14 000) and intact TSH (Mr 28 000; subunits crosslinked) and two at higher Mr separated by 14 000. The latter bands represented one or two subunits of TSH crosslinked to a subunit of the TSH receptor with Mr 57 000 (fat) or 60 000 (thyroid). These Mr values were reduced by trypsin treatment to 43 000 and 50 000, respectively. Analysis under nonreducing conditions showed that both fat and thyroid receptors have a second disulphide linked subunit of Mr 30 000.

Adipose Tissue↗