Pharmacologically distinct binding sites in rat brain for [ [formula omitted]]thyrotropin-releasing hormone (TRH) and [ [formula omitted]][3-methyl-histidine 2]TRH

Abstract

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 [ 3 H ][3-Me-His 2]TRH or [ 3 H ]TRH binding to rat cortical homogenates. [ 3 H ][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 [ 3 H ][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. [ 3 H ]TRH was observed to label a single population of low-affinity, high-capacity sites ( K d : 7.55±1.23 μM, N=6; B max: 3.40±0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [ 3 H ]TRH-labelled sites did not correlate with their affinities for [ 3 H ][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, [ 3 H ]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.