Abstract
Previous studies (Bulant, M., Delfour, A., Vaudry, H., and Nicolas, P. (1988) J. Biol. Chem. 263, 17189-17196; Bulant, M., Roussel, J. P., Astier, H., Nicolas, P., and Vaudry, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4439-4443) have shown that post-translational processing of rat thyrotropin-releasing hormone prohormone (pro-TRH) generates, besides thyrotropin-releasing hormone (TRH), a connecting decapeptide corresponding to prepro-TRH-(160-169), i.e. Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr. This peptide, which is named TRH-potentiating peptide (Ps4), is co-localized with TRH in the median eminence nerve endings and is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo. To characterize the receptor(s) for TRH-potentiating peptide in the pituitary, a highly potent and metabolically stable derivative of Ps4, [I-Tyr0]Ps4, was radioiodinated. Binding of [125I-Tyr-0]Ps4 to rat pituitary membrane homogenates was specific, saturable, reversible, and linear with membrane protein concentration. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high affinity binding sites (Kd = 0.22 nM; Bmax = 517 fmol/mg of membrane proteins). Several naturally occurring neuropeptides and hormones, including TRH, did not compete with [125I-Tyr0]Ps4 in the binding, which suggests the binding sites are specific to Ps4. Using C-terminal deletion analogs of [Tyr0]Ps4, we further showed the critical role the C-terminal residues Thr10, Val9, and Asp8 play in conferring high binding affinity and selectivity. Binding site tissue distribution and cross-reactivity binding studies suggest that the action of TRH-potentiating peptide is mediated through interaction with a specific pituitary cell-surface receptor which differ from those for TRH. [I-Tyr0]Ps4 reported in this paper, through its high binding affinity and specificity, its very low nonspecific binding, its high resistance to enzymatic degradation, and its high potentiating action in vitro should allow further progress in understanding the in vivo physiological function of Ps4.
Highlights
U.S A. 87, 4439-4443) have shown that post-trans- thyrotropinhormone secretionfrom pituitary cells, ithas lational processing of rat thyrotropin-releasing hor- been sinceidentifiedinseveral extrahypothalamicbrain mone prohormone generates, besides thy- structures and the gastrointestinal tract of mammals [3]
Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-ThrT.his which contains multiple separate copies of the TRHprogenpeptide,whichisnamedTRH-potentiatingpeptide itor sequence, Gln-His-Pro-Gly, each T R H sequence being ( P s ~ )i,s co-localized with TRH in tmheedian eminence flanked by paired basic amino acid cleavage sites and linked nerve endings and is involved in potentiation of the together by connecting fragments (Fig. 1) [4,5,6,7,8,9]
Val’, and Asp’ play in conferring high binding affinitiyngs are consistent with theview that, in the hypothalamus, and selectivity.Bindingsitetissuedistributionand all paired basic residues of pro-TRH are susceptible cross-reactivity binding studiessuggest that theaction cleavage sites for specific endoproteases presentin this tissue
Summary
Tyr were protected by thet-butyl group.After treatment of the peptidyl-resin withtrifluoroaceticacid and ether extraction, crude peptides were purified by preparativeHPLCon a WatersRCM compact preparative cartridge module (25 mm X 10 cm) eluted a t a Identification of Specific Binding Sites for TRH-potentiating. Peptide intheRatPituitaryMembranes-The saturation isotherm of the specific binding of ['251-Tyro]Ps4in rat pituiflow rate of 8 ml/min by a 0-60% linear gradient of acetonitrile in tary membrane homogenates(0.12 mg of membrane proteins/. Specific binding was saturable with increasing concen-['251-Tyro]Ps4(200,000 cpm; 0.23 nM) binding to rat pituitary trations of ['251-Tyro]Ps4(Fig. 3, inset) and was detected even membranereceptors (0.03 mg/ml membraneproteins). Within the range of concentrations studied, Nonspecific binding was determined in the presenceof a 500the specific binding of ['251-Tyro]Ps4represented 76 to 92% fold excess of unlabeled [I-Tyro]Ps4 and represented7.5% of of the value of the total bound ligand while the linear non- the totalbinding. After 120min, almost80% of the radioiodinated peptide saturation binding isotherm was linear (Fig. 3)
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