Functional and structural characterization of a dense core secretory granule sorting domain from the PC1/3 protease

Abstract

Several peptide hormones are initially synthesized as inactive precursors. It is only on entry of these prohormones and their processing proteases into dense core secretory granules (DCSGs) that the precursors are cleaved to generate their active forms. Prohormone convertase (PC)1/3 is a processing protease that is targeted to DCSGs. The signal for targeting PC1/3 to DCSGs resides in its carboxy-terminal tail (PC1/3(617-753)), where 3 regions (PC1/3(617-625), PC1/3(665-682), and PC1/3(711-753)) are known to aid in sorting and membrane association. In this article, we have determined a high-resolution structure of the extreme carboxy-terminal sorting domain, PC1/3(711-753) in micelles by NMR spectroscopy. PC1/3(711-753) contains 2 alpha helices located between residues 722-728 and 738-750. Functional assays demonstrate that the second helix (PC1/3(738-750)) is necessary and sufficient to target a constitutively secreted protein to granules, and that L(745) anchors a hydrophobic patch that is critical for sorting. Also, we demonstrate that calcium binding by the second helix of PC1/3(711-753) promotes aggregation of the domain via the hydrophobic patch centered on L(745). These results provide a structure-function analysis of a DCSG-sorting domain, and reveal the importance of a hydrophobic patch and calcium binding in controlling the sorting of proteins containing alpha helices to DCSGs.

Fig. 1.

Structure of PC1/3_711–753 in CHAPS. The 20 lowest-energy conformers were superimposed using the backbone atoms C′, C^α, and N of the first helix between residue S⁷²² and residue F⁷²⁸ (A) and the second helix between residue D⁷³⁸ and residue N⁷⁵⁰ (B). Ribbon (C and E) and helical wheel (D and F) representations of the 2 alpha helices in the PC1/3_711–753 DCSG-sorting domain. Hydrophobic side chains are shown in the ribbon representations, and hydrophobic amino acids are highlighted in orange in the helical wheels while hydrophilic amino acids are represented in blue.

Fig. 2.

PC1/3_711–753 interacts with calcium. (A) Overlay of the 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled PC1/3_711–753 in the free form (black) and in the presence of 10 mM CaCl₂ (red). Spectra were recorded in 20 mM d-11 Tris (pH = 6.5) at 26.6 °C with a protein concentration of 1.0 mM in 20 mM CHAPS. Examples of shifted signals are circled. (B) Histogram of the variations Δδ_(ppm) = [(0.17ΔN_H)² + (ΔH_N) ²]^1/2 (39).

Fig. 3.

Functional assay of the two helices in PC1/3_711–753. (A) Schematic diagram of PC1/3 fusion protein fragments used to test for DCSG sorting potential. Proteins are composed of either an Ig fragment (Fc), or Fc fused to PC1/3 peptides containing either both helices (FcPC1/3_711–753), the first helix and linker region (FcPC1_711–738) or the second helix (FcPC1/3_738–753). (B) Representative pulse–chase assay for regulated secretion of fusion proteins in AtT-20 cells are shown. For procedure see materials and methods section. Parallel wells of cells are chased for 16 h (C, chase) either in the absence (−F) or in the presence (+F) of the secretagogue forskolin. The relative efficiency of sorting is determined by comparing the amount of protein released by the secretagogue to the amount secreted constitutively in the overnight chase (+F/C). (C) Autoradiograms similar to those shown in panel A were exposed to storage phosphor screen and quantified. Shown are the ratios (mean ± SEM) of fusion protein content in the regulated (+F) versus the corresponding overnight chase (C) incubations. n = 4–6 independent transfections. ***, P < 0.001 as compared with Fc alone.

Fig. 4.

Site-directed mutagenesis of the second helix of PC1/3_711–753. Alanine substitution was used to identify residues in the second helix that are functionally important for DCSG sorting. (A) AtT-20 cells transfected with the designated alanine-substituted Fc fusion proteins were pulse-labeled with 35-S methionine. After a 16 h chase in unlabeled medium, radiolabeled fusion protein distribution was determined by immunoprecipitation. Shown are the ratios (mean ± SEM) of radiolabeled fusion protein content in cell lysates versus the corresponding overnight chase medium. (B) Parallel wells of pulse-labeled transfected AtT-20 cells were tested for regulated secretion of the indicated fusion proteins after a 16 h chase as described in the legend to Fig. 3. (C) Shown are the ratios (mean ± SEM) of fusion protein content in the regulated (+F) versus the corresponding overnight chase (C) incubations. n = 4–8, independent transfections. ***, P < 0.001; **, P < 0.01; *, P < 0.05 versus FcPC1/3_711–753.