Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone

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.

FIGURE 1.

Model of rat proTRH post-translational processing. This illustration depicts the 255-amino acid sequence of the rat 29-kDa preproTRH polypeptide. The signal sequence is cleaved upon delivery into the ER yielding the proTRH prohormone. The initial processing cleavage of proTRH begins at the TGN level by the action of PC1/3 generating an N-terminal and C-terminal intermediate forms. The numbers below the preproTRH sequence represent the location of pair of basic residues sites where PC1/3 and PC2 produce their enzymatic cleavages. After the generation of the TRH progenitor sequence (black rectangles), primarily carboxypeptidase E (CPE) and, secondarily, carboxypeptidase D (CPD) remove the pair of basic residues at the C-terminal side (17). Gln-His-Pro-Gly is then amidated by the action of peptidylglycine α-amidating monooxygenase (PAM), which uses the C-terminal Gly as the amide donor, and the Gln residue undergoes cyclization to a pGlu residue to yield TRH (11). Peptides are indicated as pXYZ nomenclature, where p indicates peptide, X is the first amino acid of each peptide, Y is the last amino acid of each peptide, and Z indicates the total of amino acids in that given peptide. On the top of the intact preproTRH (29 kDa) are shown the N-terminal (amino acids 31–52) and C-terminal (amino acids 241–255) sequences deleted for this study. Mutated residues within the N-terminal sequence are in bold. ISGs, immature SGs.

FIGURE 2.

Effect of the N-terminal preproTRH(31–52) and C-terminal preproTRH(241–255) sequence deletion on precursor protein levels in AtT20 cells under steady state conditions. A, Western blot analysis of extracted peptides from AtT20 cells transfected with wild type (WT) proTRH, Δ31–52, or Δ241–255 mutants. After 48 h of transfection, levels of wild type (26 kDa) and mutated (∼23–24 kDa) prohormone levels were analyzed by densitometric analysis of the Western blot signals (n = 4, ±S.E.). Student's t test was done to compare each with wild type (*, p < 0.05). B, proTRH gene expression analysis. Transcript levels of wild type and Δ31–52 under steady state conditions were assayed by reverse transcription-PCR (n = 3; ± S.E.). Δ31–52 mRNA was transcript as much efficiently as than wild type. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, proTRH gene translation analysis. Protein translation for wild type and mutant Δ31–52 was performed using an in vitro cell free translation system (see “Experimental Procedures”). After protein immunoprecipitation,³H-labeled translation products were separated onto 12% SDS-PAGE polyacrylamide gels. The gels were sliced, and the cpm for each slice was counted. The peaks depicted in the graph represent the amount of prohormone (26 kDa) translated. Equal amount of protein was observed in both wild type and Δ31–52 (n = 4, ±S.E.). NC represents none DNA on the reaction.

FIGURE 3.

Analysis of Δ31–52 prohormone degradation by proteasome and lysosome systems. AtT20 cells transfected with wild type proTRH or Δ31–52 were treated with vehicle solution or proteasome inhibitor lactacystin (5 μm) for 16 h (A) or BFA (5 μg/ml) for 4 h (B). Equal amounts of protein from each sample were immunoblotted with anti-pCC10 antibody that recognizes the entire precursor protein (26 kDa for wild type (WT) and 23 kDa for Δ31–52 mutant) or anti-β-tubulin. NC represents AtT20 cells transfected with pEGFP-N1 plasmid as negative control. Protein levels were quantitated by AlphaEraseFC software. Graphics show the ratio between cells treated/untreated in wild type and mutant Δ31–52. Prohormone levels of Δ31–52 are recovered ∼2-fold by proteasomal inhibitor and BFA treatment (n = 4; ±S.E.). A representative blot from a single experiment is shown.

FIGURE 4.

Effect of specific mutations within the N-terminal proregion on protein abundance under steady state conditions in ATt20 and GH4C1. Wild type (WT) and PGL mutant DNAs were transfected into AtT20 (A) and GH4C1 (C) cells. NC indicates cells transfected with pEGFP-N1 plasmid. 48 h after transfection cells were lysed for immunoblotting with anti-pCC10. This antibody recognizes the precursor (∼26 kDa for ΔPGL, PGL/EKA, PGL/GGG, and PGL/AAA ∼23 kDa for Δ31–52), but it also recognizes N-terminal intermediate forms of the precursor (15 and 9.5 kDa). Precursor protein levels (26 kDa) are expressed as percentages of the levels in wild type cells. Values represent the mean ± S.E., n = 3. *, p < 0.05 relative to wild type by Student's t test. B, wild type, AVT, and R51G/R52G mutant DNAs were transfected into AtT20 cells, and the precursor protein levels were analyzed as described above (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. NC, negative control.

FIGURE 5.

Analysis of proTRH-derived peptides secretion in wild type and N-terminal mutants. N-terminal (pEH24) (A), C-terminal (5.4 kDa) (B), and TRH (C) proTRH derived peptides from 1-h basal release (BR), 1-h stimulated release (SR) with 1 mm BaCl₂, and cell content (CC) were measured by radioimmunoassay in AtT20 cell expressing wild type (WT) and mutants Δ31–52, PGL/EKA, ΔPGL, PGL/GGG, PGL/AAA, R51G/R52G, and AVT/GGG. Peptides levels in each fraction are expressed as percentage of the levels in wild type cells (100%) (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. See Table 1 for a description of mutants.

FIGURE 6.

Effect of proteasome inhibition in wild type (WT), ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type preproTRH were exposed to vehicle solution (–) or 5 μm lactacystin (+) for 16 h. NC represents AtT20 cells transfected with pEGFP-N1 plasmid. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered is expressed as the ratio lactacystin+/lactacystin–. Inhibition of the proteasome induced 2-fold recovered ΔPGL precursor compared with wild type (A) but was 1.3-fold lower for PGL/GGG precursor (n = 4, ±S.E.). NC, negative control.

FIGURE 7.

Effect of ER transport with Brefeldin A in wild type, ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type (WT) preproTRH were exposed to vehicle solution (–) or 5 μg/ml BFA (+) for 4 h. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered was expressed as the ratio BFA+/BFA–. For 4 h of incubation, BFA proportionally induces an equal accumulation in wild type and both ΔPGL and PGL/GGG precursors (n = 4, ±S.E.).

FIGURE 8.

Determination of wild type proTRH, ΔPGL, and PGL/GGG precursor half-lives by pulse-chase experiments. 48 h after AtT20 cells transfection with wild type (WT), ΔPGL, and PGL/GGG DNA constructs, protein precursors were radiolabeled for 2 h with [³H]leucine, and protein levels (cpm) were followed at 0-, 45-, and 90-min chase times. Graphics show the evolution of proTRH precursor (26 kDa) through 90 min. The results are the means ± S.E. (n = 3).