0013-7227/90/1274-1978$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 4 Printed in U.S.A.

Processing of Thyrotropin-Releasing Hormone (TRH) Prohormone in the Rat Olfactory Bulb Generates Novel TRH-Related Peptides* MARC BULANT, JEAN CLAUDE BEAUVILLAIN, ANTOINE DELFOUR, HUBERT VAUDRY, AND PIERRE NICOLAS Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, CNRS, Universite Paris 7 (P.N., A.D., M.B.), 75251 Paris Cedex 05, France; and Groupe de Recherche en Endocrinologie Moleculaire, CNRS UA 650, Unite Affiliee a I'INSERM, Universite de Rouen (M.B., H. V.), 76134 Mont-Saint-Aignan Cedex; INSERUM U 156 (J.C.B.) 59045 Lille Cedex, France

169) and prepro-TRH-( 178-199). The dissimilarity in tissue content suggests that differential processing of TRH precursor by various enzymatic pathways may act as a regulating mechanism for TRH and TRH-related activities. The cellular localization of C-terminally extended forms of TRH in rat olfactory bulb was examined by the indirect immunoperoxidase method, using antisera directed against prepro-TRH-(160-169) and prepro-TRH-( 178-199). Cell bodies and nerve fibers were detected in the glomerular and external plexiform layers of the main olfactory bulb. The presence of extended forms of TRH in interneurons and middle tufted cells of the main olfactory bulb suggests that in light of the recent biological properties described for prepro-TRH-(160-169), these peptides may act as neuromodulators for olfactory epithelium imputs or neurotransmitters within more rostrally located olfactory areas in the forebrain. {Endocrinology 127: 1978-1985,1990)

ABSTRACT. Based on the deduced amino acid sequence of rat TRH prohormone (pro-TRH), proteolytic processing of this polyprotein precursor is expected to produce, beside TRH, several other novel peptides. These peptides should correspond to connecting segments flanking the repeated TRH progenitor sequence and to various C- and/or N-terminally extended forms of TRH. The profile of the endogenous products of the TRH system was studied in rat brain using multiple RIAs coupled to molecular sieve filtration and HPLC separations. In extracts from the rat hypothalamus, TRH and two pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199) were detected in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. In the olfactory bulb, pro-TRH is processed differently, since peptides containing TRH at their N-termini, [pGlu172] preproTRH-(172-199) and [pGlu154]prepro-TRH-(154-169), were found to be major end products along with prepro-TRH-(160-

R

AT TRH precursor (pro-TRH) contains five separate copies of the TRH progenitor sequence GlnHis-Pro-Gly, each flanked by the typical prohormoneprocessing signal, Lys-Arg at the amino end and LysArg or Arg-Arg at the carboxyl end (1) (Fig. 1). Proteolytic processing of pro-TRH at all paired basic residues is expected to yield five copies of TRH along with seven novel non-TRH peptides. These peptides should correspond to the leading and trailing pro-TRH peptide sequences and to connecting segments flanking the repeated TRH progenitor sequences. We have recently demonstrated the presence, in rat hypothalamus and spinal cord, of two pro-TRH connecting peptides, i.e. Received February 12,1990. Address all correspondence and requests for reprints to: Dr. Pierre Nicolas, Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Universite Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France. * This work was supported in part by funds from the Foundation pour la Rechereche Medicale Francaise and the Ministere de la Recherche et de l'Enseignement Superieur (87/H/0303).

prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178199) (Ps5), in addition to TRH as predominant storage forms of the TRH prohormone (2-4). Another pro-TRH connecting peptide, prepro-TRH-(83-106) has also been detected and characterized in rat hypothalamic tissues (5). Ps4, which is secreted with TRH in the rat median eminence in vitro, binds to specific sites in the distal part of the pituitary and potentiates TRH-evoked TSH secretion in vitro (6). This observation raises the interesting possibility that some of the non-TRH peptides derived from the pro-TRH sequence may exert biological activity. On the other hand, depending on the proteolytic cleavages that occur, a different pattern of pro-TRHrelated peptides, including N- and C-terminally extended forms of TRH, could be generated by partial processing of the prohormone. In light of the biological properties described for pS4, differential processing of the TRH precursor by various enzymatic pathways in various tissues may act as a regulating mechanism for TRH and/ or TRH-related activities. Therefore, elucidation of the

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PROCESSING OF PRO-TRH IN OLFACTORY LOBES

1979

pGlu-Hls-Pro-Gly-Arg-Arg-Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-VBl-Thr,-. Ser-Phe-Pro-Trp-Het-Glu-Ser-ABp-VBl-Thr.,. tfaU

(TRH-Pa

Processing of thyrotropin-releasing hormone (TRH) prohormone in the rat olfactory bulb generates novel TRH-related peptides.

Based on the deduced amino acid sequence of rat TRH prohormone (pro-TRH), proteolytic processing of this polyprotein precursor is expected to produce,...
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