Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone.
An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5.
Brady
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Laboratory of Toxicology, Harvard School of Public Health, Boston, MA 02115.
Tashjian
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D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010782:Photolysis; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D017981:Receptors, Neurotransmitter; D018025:Receptors, Thyrotropin-Releasing Hormone; C072274:TRH, 4-azidosalicylamide-; D013973:Thyrotropin-Releasing Hormone