Pharmacologically distinct binding sites in rat brain for [3H]thyrotropin-releasing hormone (TRH) and [3H][3-methyl-histidine(2)]TRH.
We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [3H][3-Me-His(2)]TRH or [3H]TRH binding to rat cortical homogenates. [3H][3-Me-His(2)]TRH was observed to bind to a single population of high-affinity, low-capacity sites (K(d): 4.54+/-0.62 nM, N=5; B(max): 4.38+/-0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K(i) of 22 nM. K(i) values for the library peptides at [3H][3-Me-His(2)]TRH-labelled sites varied from 10(-3) to 10(-9)M; the potency order was: [3-Me-His(2)]>His>Thi>Leu,Phe,Asn>Gln, Arg, Thr, Ala, HomoPhe. All other replacements had K(i) values >10(-4)M. [3H]TRH was observed to label a single population of low-affinity, high-capacity sites (K(d): 7.55+/-1.23 microM, N=6; B(max): 3.40+/-0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [3H]TRH-labelled sites did not correlate with their affinities for [3H][3-Me-His(2)]TRH-labelled sites (r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme (r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [3H]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.
Department of Biochemistry, Trinity College Dublin, Ireland. kellyja@tcd.ie
Julie A
JA
Kelly
Gillian R
GR
Slator
Kathy M
KM
O'Boyle
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008297:Male; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D017207:Rats, Sprague-Dawley; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium
D014316:Tritium; C014109:thyrotropin-releasing hormone, 3-Me-; D013973:Thyrotropin-Releasing Hormone; D011761:Pyrrolidonecarboxylic Acid