Photo‐affinity labelling of the thyrotropin receptor

Buckland, Paul Robert; Rickards, Carole R.; Howells, Rogers D.; Jones, E. Davies; Smith, Bernard Rees

doi:10.1016/0014-5793(82)80176-2

Cited by 61 publications

(36 citation statements)

References 7 publications

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“…These bands had M, values of 70500 and 85500 in the case of guinea pig fat and 74000 and 88000 in the case of guinea pig thyroid soluble receptors (table 1). A& values of 59000 and 74000 for porcine thyroid preparations have been reported in [7]. The higher M, bands were not observed when an excess of unlabelled TSH was added to the incubation mixture prior to [ fig.2).…”

Section: Resultsmentioning

confidence: 81%

“…Previous studies [7] have indicated that the two bands differing in M, by 14000 which comprise the components of the TSH-TSH receptor complexes observed on SDS-PAGE under reducing conditions represent a single subunit of the TSH receptor (designated the A subunit) cross-linked to (i) one subunit of TSH (lower A4, band) or to (ii) two subunits of TSH (higher M, band) where the TSH subunits are themselves cross-linked so dissociation cannot occur.…”

Section: Discussionmentioning

confidence: 99%

“…Highly purified bovine TSH (70 MRC units/mg) was coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) as in [7] to give a preparation which contained, on average, one molecule of HSAB on each of the 2 subunits of TSH. The HSAB-TSH retained about 40% of its TSH receptor binding and thyroid stimulating activities.…”

Section: Preparation Of '251-labelled Hsab-tshmentioning

confidence: 99%

“…]HSAB-TSH and TSH receptors (cell membranes or detergent solubilised) were incubated together, photolysed, and bound and free [1251]HSAB-TSH were separated by precipitation with polyethylene glycol in the case of soluble receptors or centrifugation in the case of cell membranes [7]. About 40% of the TSH specifically bound to the receptor was crosslinked to its receptor as judged by the inability of 2 M NaCl or sodium dodecyl sulphate (SDS) to cause dissociation of the 1251-labelled TSH-TSH receptor complex.…”

Section: Preparation and Analysis Of Crosslinked Tsh-tsh Receptor Commentioning

confidence: 99%

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

Buckland

Smith

1984

FEBS Letters

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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 (M r 14 000) and intact TSH (M r 28 000; subunits crosslinked) and two at higher M r separated by 14 000. The latter bands represented one or two subunits of TSH crosslinked to a subunit of the TSH receptor with M r 57 000 (fat) or 60 000 (thyroid). These M r values were reduced by trypsin treatment to 43 000 and 50 000, respectively. Analysis under non‐reducing conditions showed that both fat and thyroid receptors have a second disulphide linked subunit of M r 30 000.

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Section: Resultsmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of '251-labelled Hsab-tshmentioning

confidence: 99%

Section: Preparation and Analysis Of Crosslinked Tsh-tsh Receptor Commentioning

confidence: 99%

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

Buckland

Smith

1984

FEBS Letters

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“…Cross-linking analysis of the CGRP-receptor complex in intact placental membrane preparations or soluble receptor preparations was performed using ' 251-a-CGRP and the homolifunctional cross-linking disuccinimidyl suberate (DSS) essentially as described by Buckland et al [34]. Reaction conditions were as described for receptor binding, except that 50 mM Hepes pH 7.6 was substituted for Tris in buffer A.…”

Section: Cross-linking Analysesmentioning

confidence: 99%

Isolation and characterisation of a human calcitonin‐gene‐related‐peptide receptor

Foord¹,

Craig²

1987

European Journal of Biochemistry

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We describe the identification and purification of a receptor for calcitonin-gene-related peptide (CGRP) from human term placenta, using lectin and P-CGRP -Affigel affinity chromatography. The membrane-bound receptor has an estimated M , of 240000, as determined by cross-linking 1251-labelled a-CGRP (1251-a-CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263 000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross-linking studies with disuccinimidyl suberate and N-hydroxysuccinimidyl-4-azidobenzoate using membrane-solubilised, partially purified and CGRP-affinity-purified preparations, show a number of '251-or-CGRP binding subunit(s) of M , 62000 -68000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower M,. The receptor binds human fl-CGRP with greater affinity than a-CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of lZ51-a-CGRP binding to the membrane-bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP-binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.Studies on the structure and expression of the rat and human calcitonin genes have demonstrated the generation by RNA processing of alternative mRNA species from a single gene in a tissue-specific manner [l -41. The mRNA species produced encode polyproteins cleaved by post-translational events to yield either calcitonin, the major gene product in the thyroid, or a predicted 37-amino-acid amidated peptide, the calcitonin-gene-related peptide (a-CGRP). A second gene encoding a similar peptide (P-CGRP) has also been identified in rat and man [2 -61. Immunohistochemical studies using antisera raised against synthetic CGRP peptides have revealed the presence of CGRP-like material in discrete areas of the brain, and in extensive networks of sensory nerves, in particular nerves supplying the vasculature [7 -111. Elevated human plasma CGRP levels have also been documented in medullary thyroid carcinoma and lung carcinoma [3,4,12

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Thyrotropin Receptor Purification

Leedman¹,

Harrison²

1990

Receptor Purification

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Photo‐affinity labelling of the thyrotropin receptor

Abstract: Thyrotropin-thyrotropin receptor complexes Disulphide-linked subunit (SDS-polyacrylamide gel electrophoresis)

Cited by 61 publications

References 7 publications

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

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

Isolation and characterisation of a human calcitonin‐gene‐related‐peptide receptor

Thyrotropin Receptor Purification

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