Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

Amparo Romero,Isin Çakir,Charles A Vaslet,Ronald C Stuart,Omar Lansari,Hector A Lucero,Eduardo A Nillni

doi:10.1074/jbc.m803413200

Amparo Romero, Isin Çakir + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m803413200

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Abstract

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

Highlights

Hypophysiotropic thyrotropin-releasing hormone (TRH)2 is produced in the paraventricular nucleus of the hypothalamus and stimulates thyroid-stimulating hormone secretion from the anterior pituitary [1]
PreproTRH241–255 preproTRH40–42 preproTRH40–42 preproTRH40–42 preproTRH34–36 preproTRH34–36 preproTRH51,52 preproTRH40–42 leaving intact the first 6 amino acids preceded by the signal sequence Deletion of C-terminal 15 amino acids Pro-Gly-Leu replaced by Glu-Lys-Ala Deletion of Pro-Gly-Leu Pro-Gly-Leu replaced by Gly-Gly-Gly Ala-Val-Thr replaced by Gly-Gly-Gly Deletion of Ala-Val-Thr Arg-Arg replaced by Gly-Gly Pro-Gly-Leu replaced by Ala-Ala-Ala pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium supplemented with high glucose, 30 mM sodium bicarbonate, 1 mM sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO2 as previously described [20]
A typical double band was observed for proTRH when using this specific antibody that could represent a potential glycosylation of proTRH

Summary

EXPERIMENTAL PROCEDURES

EcoRI digestion and subcloned into pCDNA3.1/zeoϩ plasmid (Invitrogen). This plasmid drives expression of cloned genes with the constitutively active cytomegalovi-. After the generation of the TRH progenitor sequence (black rectangles), primarily carboxypepfor creation of eight different pretidase E (CPE) and, secondarily, carboxypeptidase D (CPD) remove the pair of basic residues at the C-terminal proTRH mutants. ⌬241–255 PGL/EKA ⌬PGL PGL/GGG AVT/GGG ⌬AVT R51G/R52G PGL/AAA preproTRH241–255 preproTRH40–42 preproTRH40–42 preproTRH40–42 preproTRH34–36 preproTRH34–36 preproTRH51,52 preproTRH40–42 leaving intact the first 6 amino acids preceded by the signal sequence Deletion of C-terminal 15 amino acids Pro-Gly-Leu replaced by Glu-Lys-Ala Deletion of Pro-Gly-Leu Pro-Gly-Leu replaced by Gly-Gly-Gly Ala-Val-Thr replaced by Gly-Gly-Gly Deletion of Ala-Val-Thr Arg-Arg replaced by Gly-Gly Pro-Gly-Leu replaced by Ala-Ala-Ala pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium supplemented with high glucose, 30 mM sodium bicarbonate, 1 mM sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO2 as previously described [20].

Moieties recognized

RESULTS

DISCUSSION