J. Biol. Chem., Vol. 267, Issue 36, 25697-25702, Dec, 1992
Characterization of receptors for thyrotropin-releasing hormone- potentiating peptide on rat anterior pituitary membranes
A Ladram, M Bulant and P Nicolas
Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Universite, Paris 7, France.
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.
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