Disruption of a Receptor-Mediated Mechanism for Intracellular Sorting of Proinsulin in Familial Hyperproinsulinemia
Savita Dhanvantari,
Fu-Sheng Shen,
Tiffany Adams,
Christopher R. Snell,
ChunFa Zhang,
Robert B. Mackin,
Stephen J. Morris and
Y. Peng Loh
Section on Cellular Neurobiology (S.D., F.-S.S., T.A., C.Z., Y.P.L.), Laboratory of Developmental Neurobiology, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4480; Medivir U.K. Ltd. (C.R.S.), Cambridge CB1 9PT, United Kingdom; Creighton University School of Medicine (R.B.M.), Omaha, Nebraska 68178-0306; and Montreal Neurological Institute (S.J.M.), McGill University, Montreal, Quebec, Canada H3A 2B4
Address all correspondence and requests for reprints to: Y. Peng Loh, National Institutes of Health, Building 49, Room 5A38, MSC 4480, Bethesda, Maryland 20892-4480. E-mail: lohp{at}mail.nih.gov.
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ABSTRACT
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In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a ß-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.
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INTRODUCTION
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ABNORMAL ELEVATION OF serum mutant proinsulin levels is a hallmark of familial hyperproinsulinemia. Specific mutations in the proinsulin gene are the only known cause of the disease, suggesting that changes in proinsulin structure can trigger abnormally high secretion of unprocessed and partially processed mutant proinsulin from the ß-cell of the pancreas. Four point mutations associated with seven kindreds with hyperproinsulinemia have been reported. One family had a substitution of His10 to Asp on the B chain of proinsulin (known as B10) (1). The other families all had point mutations resulting in a substitution at Arg65, the C peptide-A chain junction processing site, to either His (2), Pro (3), or Leu (4). To model this syndrome, the trafficking and secretion of B10 proinsulin have previously been examined in a cell line (5) and a transgenic mouse (6) harboring the mutated proinsulin gene. These two studies showed that B10 proinsulin is largely not processed to insulin and is secreted in an unregulated manner, demonstrating the utility of cell and animal models in determining the basis of familial hyperproinsulinemia. While it is clear that the B10 mutation of proinsulin leads to unregulated secretion, the exact molecular and cellular mechanism affecting the secretory process in this disease has as yet not been established.
We hypothesized that alterations in the molecular structure of proinsulin in familial hyperproinsulinemia cause a disruption in the mechanism of sorting to the regulated secretory pathway (RSP). After synthesis in the endoplasmic reticulum (ER), proinsulin is transported to the trans-Golgi network (TGN). We have previously proposed that proinsulin is sorted by a signal receptor-mediated mechanism at the TGN and packaged into immature regulated secretory granules in an neuroendocrine cell line (7, 8, 9). Sorting may also occur from the immature secretory granule in ß-cells, whereby proinsulin is retained within the granules and other proteins are secreted in a constitutive-like manner (10). Proinsulin is then converted within immature secretory granules into insulin and C-peptide (11, 12), and insulin is then stored within mature secretory granules until released upon stimulation. Missorting of mutant proinsulin, perhaps due to a lack of binding to its sorting receptor, would therefore lead to unregulated or constitutive release of unprocessed proinsulin.
Here we show that proinsulin contains a specific three-dimensional molecular sorting signal within the insulin domain that interacts with a sorting receptor, membrane carboxypeptidase E (CPE). When this interaction is disrupted in a ß-cell line, either through mutation of the sorting signal or through depletion of CPE, proinsulin is missorted and constitutively secreted. The mutations found in familial hyperproinsulinemia disrupt the binding of mutant proinsulin to CPE, resulting in inefficient sorting of proinsulin to the regulated secretory pathway.
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RESULTS
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Identification of a Putative RSP Sorting Signal on Proinsulin by Molecular Modeling
AnRSP sorting signal was predicted to reside in the insulin region of proinsulin (Fig. 1A
) because sorting to the regulated secretory pathway is maintained even when the C-peptide was removed (13), and the insulin structure within proinsulin is identical to the x-ray crystal structure of free insulin (14). We found a motif comprising EB13 LB17 LA16 EA17 (Fig. 1B
), which is similar to the three-dimensional sorting signal previously proposed for the targeting of proopiomelanocortin (POMC) (9, 15). This molecular motif (Fig. 1B
) should allow monomeric proinsulin to interact with the basic residues (R255, K260) in the binding domain of the proposed sorting receptor, membrane CPE, via the acidic side-chains (9). However, hexamerization of proinsulin, which may begin at the TGN, renders the EB13 residue inaccessible for binding a sorting receptor (Fig. 1C
) (16). In the insulin hexamer, the EA17 residues from the A chains of two adjacent insulin dimers may function as the sorting signal because they have similar distance geometry to that of the EB13 LB17 LA16 EA17 motif in the monomer (Fig. 1C
). Therefore, the predicted sorting signal motif can accommodate both the monomeric and hexameric forms of proinsulin.

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Fig. 1. Molecular Modeling of Proinsulin
A, Diagrammatic structure of proinsulin showing A and B chains and the linking C-peptide. B, The predicted signal, EB13 LB17 LA16 EA17, involved in sorting the insulin monomer to the RSP. C, The predicted RSP sorting signal in the insulin hexamer is composed of, LA16 and EA17 from two adjacent dimers. The structures are from the x-ray crystallographic structure for the insulin hexamer, PDB identification no. 1ZNJ. The leucine sidechains are shown as green space-fill atoms and the acidic residues as red/green space-fill atoms. The acidic residues in the sorting signals with the appropriate inter-ß carbon distance for docking to the proposed receptor, CPE (see Materials and Methods), are indicated by the arrows. The backbones are shown as ribbon traces. E, Glu; L, Leu.
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Confirmation of the Model by Site-Directed Mutagenesis
We mutated each of the four residues within the predicted motif (Fig. 1B
) in various combinations and transfected them into AtT-20 cells, a mouse pituitary cell line that expresses endogenous CPE. For the experiments shown in Fig. 2
, two sorting signal mutants of proinsulin were used: one in which the two A chain residues were mutated (EB13 LB17 SA16 AA17, termed ELSA, where S = serine and A = alanine), and one in which the two B chain residues were mutated (AB13 SB17 LA16 EA17, termed ASLE).

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Fig. 2. Confirmation of the Model by Site-Directed Mutagenesis
A, The effect of high K+ stimulation on the release of WT and mutant insulins. Bar graphs show the % of total (cells + media) immunoreactive WT and mutant insulins ELSA and ASLE secreted under basal (white bars) and stimulated (black bars) conditions. Values represent the mean ± SEM; n = 3 for three experiments. *, P < 0.05; **, P < 0.01 compared with unstimulated basal release of WT proinsulin. B, Western blot analysis of constitutive secretion of WT and ASLE and ELSA mutant proinsulins over a 2-h period. C, Immunocytochemical localization of WT and mutant proinsulin/insulin. AtT20 cells were transfected with WT (ac) or mutant proinsulins, ELSA (df) and ASLE (gi). Proinsulin/insulin appears green and colocalization with TGN38 appears yellow/orange (c, f, and i). Arrows indicate punctate granules. Arrowheads indicate insulin/TGN-38 double staining in the TGN. Scale bars, 10 µM.
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To determine sorting of these proinsulin mutants to the regulated secretory pathway, stimulated and constitutive secretion of the mutant proinsulins were analyzed by RIA and Western blotting, respectively. In addition, the subcellular distribution of insulin-like immunoreactivity (ILI) was visualized by fluorescence immunocytochemistry. In AtT-20 cells transfected with wild-type (WT) proinsulin, we observed a 2.3-fold increase in ILI upon stimulation with 50 mM K+ (Fig. 2A
, WT). As expected for AtT-20 cells, there was also some basal constitutive secretion of transfected proinsulin, as reported by others (Fig. 2B
) (13, 17). Punctate immunofluorescent staining of ILI was evident along and at the tips of the processes (Fig. 2C
, a and b, small arrows), characteristic of subcellular localization in the granules of the regulated secretory pathway. Double label experiments with TGN38, a TGN marker shows presence of proinsulin in the TGN as well (Fig. 2C
, c, thick arrow showing yellow staining).
In contrast, when the portion of the sorting signal residing in the A chain of proinsulin was mutated, (mutant ELSA), this mutant was constitutively secreted, as reflected by the significantly higher basal release compared with WT proinsulin and lack of secretory response to high K+ (Fig. 2A
, ELSA). Western blotting analysis of medium collected over a 2-h period from cells transfected with mutant ELSA further demonstrated higher basal (constitutive) secretion of this mutant proinsulin relative to WT proinsulin (Fig. 2B
). This mutant proinsulin showed only a perinuclear immunostaining distribution (Fig. 2C
, d and e) that overlapped with that of TGN38, (Fig. 2C
, f, thick arrow showing orange staining), indicating that the mutant proinsulin was transported out of the ER cisternae to the TGN, but not to secretory granules. Thus, mutation of LA16 EA17 on the A-chain of proinsulin leads to apparent complete misrouting of insulin to the constitutive pathway.
Interestingly, mutation of the sorting signal residues on the B chain (termed ASLE) only partially disrupted sorting to the regulated secretory pathway. Secretion experiments using this mutant revealed some stimulated secretion; however, this was not statistically significant (Fig. 2A
). Western blotting of 2 h basal medium from cells expressing this ALSE mutant proinsulin showed no detectable increase in constitutive secretion of this mutant compared with WT proinsulin (Fig. 2B
) similar to that observed in Fig. 2A
. Punctate immunostaining of proinsulin was observed in the cell processes of approximately half of the 249 cells counted, although the staining was less intense than that of WT proinsulin (Fig. 2C
, gi). Only perinuclear staining that overlapped with TGN38 was evident in the rest of the cells (Fig. 2C
, i, orange staining indicated by thick arrow). An important observation was that mutations on the A chain affected proinsulin sorting to a much greater extent than did mutations on the B chain, indicating that the A chain residues may be exposed suitably to bind to CPE.
Mutation of another residue that is not a part of the sorting signal motif, SB9 to P on the B chain, had no effect on sorting, as the pattern of immunostaining and stimulated secretion were similar to that observed for WT proinsulin (data not shown). As well, mutation of the entire sorting signal (ASSA), the two acidic (ALLA) or the two hydrophobic (ESSE) residues resulted in the missorting of proinsulin as evidenced by immunocytochemical localization in the TGN (data not shown). Taken all together, the results from the site-directed mutagenesis studies support our theoretical model showing that the four residues, EB13 LB17 LA16 EA17, constitute a sorting signal necessary for targeting monomeric proinsulin to the regulated secretory pathway. In the proinsulin hexamer, the same motif can be formed by the A chain residues LA16 EA17 from two adjacent proinsulin dimers (Fig. 1C
). The lesser effect on sorting to the regulated secretory pathway when proinsulin was mutated at EB13 LB17 compared with the LA16 EA17 mutation suggests that proinsulin exists largely as hexamers at the TGN where sorting begins. This is consistent with observations that proinsulin can self associate at neutral as well as acidic pHs (18). Indeed, hexamerization provides for much greater sorting efficiency of proinsulin.
RNA Interference (RNAi) Depletes CPE and Impairs the Sorting of Proinsulin
To show that the sorting of insulin to the regulated secretory pathway in ß-cells requires the presence of a previously identified sorting receptor, CPE, we depleted CPE by treating INS-1 cells, a ß-cell line, with dsRNAs directed at the mRNA for rat CPE. Such treatment would deplete the target mRNA through RNAi (19, 20). As a control, we transfected cells with dsRNA directed against an irrelevant mRNA, GAPDH. Western blot analysis shows that RNAi decreased CPE to low levels, whereas the control dsRNA had no effect (Fig. 3A
). Secretion experiments carried out in CPE-depleted cells clearly show a constitutive pattern of secretion of (pro)insulin, with a significantly higher level of basal secretion compared with control cells (P < 0.05) and no response to secretagogue stimulation (Fig. 3A
). These results demonstrate a requirement for CPE in the sorting of proinsulin to the regulated secretory pathway in ß-cells.

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Fig. 3. Interaction with CPE Is Required for the Sorting of Insulin to the Regulated Secretory Pathway in a ß-Cell Line
A, Depletion of CPE using RNAi results in the constitutive secretion of insulin. INS-1 cells were transfected with 200 nM of a 21-oligomer dsRNA spanning nucleotides 329349 of rat CPE or with 200 nM of a 21-oligomer dsRNA spanning GAPDH (Control). Western blot analysis for CPE (top panel) and secretion experiments (bottom panel) were carried out 72 h after transfection. B, Insulin binds to CPE in vitro. Ten micrograms of secretory granule membranes were incubated with [125I]insulin in the absence (white bars) or presence (black bars) of 100 µM N-POMC1-26 for 1 h at pH 5.5 (left) or pH 6.5 (right). Membranes were collected by centrifugation and counted. Values are means ± SEM; n = 3. *, P < 0.01. C, A dominant negative mutant of CPE impairs the regulated secretion of insulin from INS-1 cells. A plasmid encoding a 26-amino acid fragment of CPE encompassing the sorting signal binding site fused to GFP (dominant negative, DN) was transfected into INS-1 cells. Secretion experiments were carried out 24 h after transfection of the DN CPE cDNA. For all secretion experiments (A and C), both cells and media were analyzed by RIA for insulin. Results are expressed as percent of total ILI released. Values are means ± SEM; n = 3. *, P < 0.05, **, P < 0.001. Statistical analysis was carried out for stimulated relative to basal release in control cells; basal release from CPE siRNA or DN CPE transfected cells relative to basal release from control cells; and stimulated relative to basal release in CPE siRNA or DN CPE transfected cells which was not significant for both experiments. Results are representative of two to four independent experiments. D, Predicted interaction between insulin and CPE. Molecular models of the sorting signal binding site on CPE and the sorting signal of proinsulin can be manually docked to visualize the interaction. On the left, EB13 and EA17 of the proinsulin monomer can be docked to R255 and K260, the sorting signal binding determinant of CPE. On the right, the EA17 residues of adjacent dimers within the proinsulin hexamer can be docked with the sorting signal binding site on CPE. R, Arg; K, Lys.
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Insulin Can Bind to CPE at an Intragranular pH
We have previously shown that proinsulin binds in an N-POMC1-26-displaceable manner to CPE in vitro (9, 21). To show that the sorting signal of proinsulin resides in the sequence of mature insulin, we carried out binding studies using pituitary secretory granule membranes as a source of membrane CPE and [125I]insulin as ligand (Fig. 3B
). Although significant binding and displacement was measured at pH 6.5 (the approximate pH of the TGN) (22), insulin seemed to bind with higher affinity at pH 5.5 (the pH of mature secretory granules) (11) with a greater degree of displacement by N-POMC1-26 (Fig. 3B
). This is noteworthy because proinsulin bound maximally to CPE at pH 6.06.5 (21).
A Dominant Negative Mutant of CPE Impairs Sorting of Proinsulin in Vivo
To corroborate our in vitro binding data, we demonstrated that proinsulin requires binding to CPE in vivo to be sorted to the regulated secretory pathway. We transfected INS-1 cells with a construct encoding a 26-residue sequence of CPE that encompasses the sorting signal binding site, R255 and K260 (9). We postulated that this sequence would behave as a dominant negative by competing with endogenous CPE for binding to proinsulin in vivo. Such binding would predict that the proinsulin would not be sorted to the RSP because the dominant negative construct lacks sorting information (23) and would be secreted constitutively. Indeed, when INS-1 cells were transfected with a plasmid encoding the dominant negative CPE, the insulin secretory response to stimulation was obliterated, and basal secretion was significantly higher than cells transfected with the trkA-green fluorescent protein (GFP) control plasmid (P < 0.05, Fig. 3C
). No effect on the regulated secretion of insulin was seen in cells transfected with the trkA-GFP control plasmid (Fig. 3C
). Taken together, the results shown in Fig. 3
provide strong evidence that proinsulin requires interaction with CPE in vivo to be targeted to the regulated secretory pathway in ß-cells. Our computer modeling analysis predicts the feasibility of docking between CPE and the insulin monomer and hexamer (Fig. 3D
). The sorting signal in the insulin monomer, EA17 and EB13, and on the hexamer, contributed by EA17 on adjacent dimers, can dock with R255 and K260, the previously identified POMC sorting signal binding site on CPE (Fig. 3D
) (9). This interaction allows for the subsequent sorting and retaining of proinsulin and insulin, respectively, in the regulated secretory pathway of ß-cells.
Mutations Causing Familial Hyperproinsulinemia Are Sorted Inefficiently to the Regulated Secretory Pathway
We generated two of the Arg65 mutations, found in familial hyperproinsulinemia, Arg65Leu and Arg65Pro and determined their subcellular trafficking in AtT-20 cells. Both mutants were secreted at a significantly higher basal level than WT proinsulin (Fig. 4A
, right) over a 2-h period. However, both mutants were secreted in a regulated manner in response to 50 mM K+ (Fig. 4A
, left). Analysis of subcellular localization by immunocytochemistry showed that, in contrast to the punctate staining pattern of WT proinsulin in the processes (Fig. 4B
, top row, arrows), both Arg65Leu (Fig. 4B
, middle row) and Arg65Pro mutants (Fig. 4B
, bottom row) colocalized largely with a Golgi marker, p115 (24). Punctate staining was observed in some of the cells expressing the proinsulin mutants (see arrows) but the most robust staining was seen in the perinuclear area, overlapping with p115 (orange staining). These results indicate that the Arg65 mutant proinsulins found in familial hyperproinsulinemia are sorted inefficiently to the regulated secretory pathway, likely due to the disruption of their ability to bind to the sorting receptor.

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Fig. 4. Proinsulin Mutants Found in Familial Hyperproinsulinemia Are Partially Missorted
A, AtT-20 cells were transfected with WT proinsulin, R65L, or R65P proinsulin and both basal (right) and stimulated secretion (left) were assessed. For basal secretion, media and cells were collected after 2 h; for stimulated release, cells were incubated in the absence or presence of 50 mM K+ for 30 min following a 2-h basal period. Both cells and media were analyzed by RIA for insulin. Results are expressed as percent of total ILI released. Values are means ± SEM; n = 3, *, P < 0.001 comparing stimulated vs. basal release for cells expressing WT, R65L or R65P (2a, left), and mutants vs. WT (2a, right). Results are representative of three independent experiments. B, Immunocytochemical localization of proinsulin and familial hyperproinsulinemia (FH) mutants. AtT-20 cells were transfected with proinsulin (WT), or FH mutants. Insulin appears green and the Golgi protein p115 marker appears red. Perinuclear staining (yellow) indicates the colocalization of proinsulin and p115 in the Golgi. Note the presence of punctate staining in the processes of cells transfected with WT proinsulin (arrow). In cells transfected with FH mutant proinsulins, some contained punctate staining in the processes and others did not (see arrows).
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Proinsulin Mutants Exhibit Impaired Binding to CPE
To determine the mechanism of the inefficient sorting of the proinsulin mutants, we carried out binding experiments to assess interaction between CPE and recombinant WT or mutant proinsulins generated from a bacterial expression system (25). Specific binding of the Arg65 proinsulin mutants to CPE was significantly decreased compared with that of WT proinsulin (P < 0.01) (Fig. 5A
). Molecular modeling studies indicate that it is feasible that the Arg65 mutation at the C-A junction could induce conformational changes to the C-peptide structure and that because the C peptide masks the majority of the exposed insulin hexamer structure (26), a change in accessibility of the EA17 residues to CPE (see Fig. 3D
) could easily occur, resulting in poorer binding to CPE. However, we must await detailed x-ray crystal structural information to fully understand the molecular basis for the decreased binding of the Arg65 proinsulin mutants to CPE.

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Fig. 5. Impaired Binding of FH Proinsulin Mutants to CPE
A, Membranes were prepared from an enriched secretory granule fraction of bovine neural lobe pituitaries. [125I]Proinsulin (WT), R65L or R65P proinsulin was incubated with 10 µg of membranes for 1 h, after which membranes were collected by centrifugation, washed and counted. Values (means ± SEM; n = 3; *, P < 0.01 relative to WT) are expressed as percent of specific binding of WT proinsulin. B, B10 proinsulin does not bind to CPE due to a disrupted sorting signal. Values (means ± SEM; n = 3; *, P < 0.05 relative to WT) are expressed as cpm of specific binding. C, Dimerization disrupts the sorting signal. The structure of the insulin dimer, PDB identification no. 4INS, shows that the EB13 and EB'13 residues are buried in the dimer interface, leaving the EA17 and EA'17 exposed. The backbone and sidechains of interest are shown as rods.
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Interestingly, B10 proinsulin showed very little specific binding to membranes expressing CPE (Fig. 5B
). It has been shown previously that B10 proinsulin preferentially forms dimers instead of hexamers (27). To explain the lack of binding of B10 proinsulin to CPE, we modeled the structure of the B10 dimer to examine the orientation of the sorting signal. Molecular modeling predicted that the sorting signal is disrupted in the dimeric form (Fig. 5C
) because, within the dimer, the EB13 residues are inaccessible for binding, and the two EA17 residues are predicted to be 27 Å apart. This distance geometry of the two acidic side-chains is not compatible with that of its corresponding binding site on CPE (Fig. 3D
) and should preclude any interaction.
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DISCUSSION
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In cases of familial hyperproinsulinemia, autosomal dominant mutations in the proinsulin gene are linked with impaired proinsulin processing and subcellular trafficking as revealed by studies of a transgenic mouse model of familial hyperproinsulinemia carrying the B10 mutation (6). To investigate the molecular and cellular basis of this disease, we have examined the sorting of normal proinsulin and various mutant proinsulins to the regulated secretory pathway in AtT-20 and ß-cells. We have previously shown that the regulated secretion of insulin was impaired in Neuro2A cells in which CPE had been depleted (8). These results indicated that CPE was required for the sorting of proinsulin, possibly by acting as a sorting receptor in a fashion similar to that of POMC (7, 9). We now provide experimental and theoretical evidence that proinsulin binds to CPE via a structural sorting signal similar to that on POMC. Importantly, we show that this interaction is required in vivo in a ß-cell line for the sorting of proinsulin to the regulated secretory pathway. We used these findings to construct a cellular model of familial hyperproinsulinemia and show that the interaction between CPE and proinsulin is disrupted in this disease.
We used molecular modeling of the insulin monomer and hexamer to screen for a structural sorting signal similar to that found in POMC (15) and tested our predictions in vivo using site-directed mutagenesis. We propose that two acidic residues, EA17 and EB13, in the insulin monomer, and EA17 from adjacent dimers in the insulin hexamer, are present in a similar distance geometry to those residues in the sorting signal of POMC, and are sufficiently exposed to allow hydrogen bonded interactions between either the insulin monomer or hexamer and a prohormone sorting receptor, CPE. Therefore, we conclude that, similar to POMC, the acidic residues in the sorting signal of proinsulin act as ligand determinants involved in binding to R255 and K260, the sorting signal binding site of CPE (9). The function of the hydrophobic residues is likely to provide the proper environment to facilitate the interaction of the acidic residues in the motif with the receptor.
The three-dimensional structure of the sorting signal on insulin indicated a possible interaction with CPE. Depletion of CPE through RNAi and transfection of a dominant negative form of CPE resulted in the constitutive secretion of insulin in a pancreatic ß-cell line. These results provide the first evidence in ß-cells that proinsulin or insulin requires interaction with the sorting signal binding site of CPE for sorting to the regulated secretory pathway. Interestingly, pancreatic islets obtained from the CPEfat/CPEfat mouse showed stimulated secretion of insulin (28). This mouse expresses a mutant form of CPE (S202P) that is enzymatically inactive and susceptible to degradation in the pituitary (29). However, Western blot analysis showed that a significant amount of immunoreactive CPE was present in the pancreatic islets (29). Moreover, this mutant CPE could exit the ER and be transported to the granules of the regulated secretory pathway in NIT3 cells derived from the ß-islets of CPEfat/CPEfat mice (30). Because we have previously shown that this mutant form of CPE binds prohormones, including proinsulin, with the same affinity as WT CPE (9), the presence of mutant CPE in islets of CPEfat/CPEfat mice (29), and the regulated secretion of insulin from these islets (28) are observations that also strongly support a role for CPE in the sorting of proinsulin. Furthermore, recent studies showing that overexpression of membrane-bound CPE in InsGH3 clones engineered to express furin-cleavable proinsulin increased regulated secretion of insulin from these cells, provide yet another observation in support of a role of CPE in proinsulin sorting to the regulated secretory pathway (31).
Our elucidation of a molecular mechanism of proinsulin sorting provides an understanding of familial hyperproinsulinemia. Our data indicate that the inefficient sorting of the B10 proinsulin mutant to the regulated secretory pathway (5, 6) is due to its lack of binding to CPE because its dimeric structure disrupts the sorting signal. We also provide evidence for the first time that a defect in the CPE-mediated sorting of proinsulin forms the molecular and cellular basis for familial hyperproinsulinemia caused by mutations at Arg65. Both Arg65Leu and Arg65Pro mutants showed poorer binding to CPE and higher basal release of insulin-like immunoreactivity but also a secretory response to stimulation. This is consistent with the phenotype of patients harboring a mutation at Arg65, who have high fasting levels of plasma insulin-like immunoreactivity but normal glucose tolerance (2); other patients have abnormal glucose tolerance, but this is not associated with this mutation (3, 4). It was postulated that the persistent fasting hyperproinsulinemia in these patients could be a result of improper processing of proinsulin and reduced plasma clearance. We now show that a third factor, impaired sorting of proinsulin due to reduced binding to CPE, also contributes to the phenotype of familial hyperproinsulinemia.
Our model (Fig. 6
) illustrates the secretory pathways to which WT and mutant proinsulin may be targeted. WT and mutant proinsulin can oligomerize (32) and bind to CPE at the TGN. However, unlike prohormones in other (neuro)endocrine cells, the sorting of proinsulin in ß-cells may occur primarily in the immature secretory granule, rather than the TGN (10). Therefore, the binding of proinsulin to CPE in the TGN may not be a necessary prerequisite for its entry to the regulated secretory pathway. Rather, processed normal insulin (Fig. 3
), or a small portion of mutant proinsulin and intermediates (Fig. 5
, A and B) can bind to CPE in the immature secretory granule and be retained in this compartment and will be available for regulated secretion elicited by glucose. The major proportion of mutant proinsulins, due to reduced binding to CPE will not be retained in the immature secretory granule, but secreted primarily unprocessed, via the constitutive-like pathway, in an unregulated manner (33). Together with the reduced clearance of proinsulin, this mechanism would then account for the high levels of mutant proinsulin in the plasma of patients with familial hyperproinsulinemia.

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Fig. 6. Proposed Mechanisms for the Sorting of Proinsulin and Proinsulin Mutants Found in Familial Hyperproinsulinemia
We hypothesize that proinsulin oligomerizes and binds to CPE, a transmembrane sorting receptor, at the TGN via its ELLE sorting signal and enters the immature secretory granule (ISG) subsequent to budding of the TGN. As the ISG gradually becomes more acidic after formation, proinsulin dissociates from CPE and is processed to insulin and C-peptide. In the lower pH environment of the ISG, mature insulin associates favorably with CPE, also through the ELLE motif, and is retained within the secretory granule. Mutant proinsulins found in familial hyperproinsulinemia (FH) show reduced binding to CPE and so may follow three secretion pathways. Mutant proinsulin that is not bound to CPE at the TGN may enter constitutive secretory vesicles (CV) and be secreted in an unregulated manner. Mutant proinsulin that enters ISG by binding to CPE at the TGN or by default (10 ) would largely be removed into constitutive-like vesicles (CLV) due to inefficient binding to CPE in the ISG, and released primarily unprocessed, but may also include partially processed forms, in an unregulated manner. The mutant proinsulin remaining in the ISG would be fully (B10 mutant) or partially (Arg65 mutants) processed and would be stored in this compartment, which eventually becomes mature secretory granules (MSG). Contents of the MSG are then secreted in a glucose-dependent manner. The inefficient sorting and retention of mutant proinsulins can account for the high levels of these molecules found in the plasma of patients with familial hyperproinsulinemia.
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In conclusion, this study has revealed the prevalence of intracellular missorting as a basis for the disease state of patients bearing different types of mutations in the proinsulin gene. In addition, with the identification of the amino acids in the regulated secretory pathway sorting signal of proinsulin, any mutations in the proinsulin gene at those residues that may be uncovered in the future, will be predicted to result in defective proinsulin sorting leading to hyperglycemia. Thus, recognition of the intracellular missorting of proinsulin as a potential cause of certain forms of diabetes will aid in the proper diagnosis and treatment of the disease.
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MATERIALS AND METHODS
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Analysis of Insulin Monomer and Hexamer Structures
The inter-ß carbon distances between all acidic residues in the x-ray structures, Brookhaven Protein Data Bank (PDB) identification no. 4INS and 1ZNJ (34, 35, 36) were calculated from the atomic coordinates. By comparison with the inter-ß carbon distances of the acidic residues, D10 and E (14) (15.8 Å), identified as the receptor binding determinants within the sorting signal for POMC (9), the only surface residues with comparable inter-ß carbon distances in the insulin monomer structure were EB13 and EA17 (14.86 Å) (Fig. 1B
), and between EA17 residues on adjacent dimers (14.87 Å) in the insulin hexamer (Fig. 1C
). Adjacent hydrophobic residues were included to match the sequence homology in the structure of the POMC sorting signal structure, i.e. [Hy].[Ac].[X]n.[Hy]. [X]3.[Ac], where Hy is an aliphatic hydrophobic amino acid, Ac is an acidic amino acid and X is any residue. In the proposed proinsulin sorting receptor, CPE, the inter-ß carbon distance between R255 and K260 residues that are essential for binding proinsulin is modeled at 12.8 Å (9).
Site-Directed Mutagenesis of Proinsulin
The human preproinsulin cDNA (a gift from K. Docherty, University of Aberdeen, Aberdeen, Scotland, UK) was subcloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) at the EcoRI site to generate proinsulin/pcDNA3.1. The correct orientation of the proinsulin insert was verified by sequencing. We generated point mutations of the residues comprising the sorting signal binding site; EB13 LB17 LA16 EA17 (Fig. 1B
), as well as the mutations implicated in familial hyperproinsulinemia, R65L and R65P, using the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA). The presence of the mutated residues was confirmed by sequencing.
Secretion Experiments in AtT-20 Cells
AtT-20 cells were transfected with WT proinsulin/pcDNA3.1 or mutants. For stimulated secretion experiments, 24 h following transfection, AtT-20 cells were incubated with serum-free DMEM without (basal), or with 50 mM KCl (stimulated) for 60 min at 37 C. Media were collected, trifluoroacetic acid added to 0.1%, and passed through a Sep-Pak C18 cartridge (Waters Corp., Milford, MA). Insulin/proinsulin was eluted with 80% acetonitrile. The cells were scraped in PBS, centrifuged, and resuspended in 10 mM Tris-HCl containing 1 mM EDTA, 0.1% deoxycholate, and 1 mM of the protease inhibitor, aminoethylbenzene sulfonyl fluoride hydrochloride (Sigma, St. Louis, MO). The suspension was freeze-thawed three times, centrifuged, and the supernatant was removed for RIA using insulin-specific and proinsulin RIA kits (Linco Research, Inc., St. Charles, MO). For constitutive secretion studies, after transfection of the cells, the medium was replaced with serum-free DMEM containing 0.25x Complete mini protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN) and the cells incubated for 2 h at 37 C. The cells and medium were collected and prepared for RIA as described above. For the secretion experiments where the basal medium was assayed by Western blotting, antiproinsulin (human) antibodies (Peninsula Laboratories, San Carlos, CA) were also included during the 2-h incubation. At the end of the incubation period, Pansorbin cells (Calbiochem-Novabiochem Corp., La Jolla, CA) were added to the collected medium and incubated for 1.5 h to precipitate the antibody-antigen complex. The Pansorbin cells were centrifuged, washed with RIPA buffer containing 0.1% sodium dodecyl sulfate and extracted for SDS-PAGE through 16% Tris-Glycine gels followed by Western blotting for proinsulin. The AtT-20 cells after the 2-h incubation were also analyzed for proinsulin by Western blot. The level of expression of the WT, ASLE, and ELSA constructs were very similar.
Immunocytochemistry
AtT-20 cells were prepared for immunocytochemistry as described previously (37) using antibodies against insulin (Peninsula) and TGN38, a protein marker for the TGN. Insulin/proinsulin was visualized with a fluorescent isothiocyanate-conjugated secondary antibody and TGN38 with a Texas Red-conjugated secondary antibody (Invitrogen). In experiments with Arg65 mutant proinsulins, cells were incubated with an anti-p115 antibody (Transduction Laboratories, Lexington, Kentucky) which marks the Golgi, along with the insulin antibody. Images were captured using a MRC-1000 confocal microscope (Bio-Rad, Hercules, CA) and analyzed using Adobe Photoshop 6.0.1 (Adobe, San Jose, CA).
CPE Dominant Negative cDNA Construct
To examine the requirement for CPE in proinsulin sorting in vivo, a dominant negative CPE construct was generated by fusion of three different sequences: the signal peptide of TrkA (receptor tyrosine kinase for nerve growth factor) was fused to a sequence of CPE that contains its sorting signal binding domain (Asn289-Thr314), which was fused in frame to GFP by cloning into the HindIII-KpnI sites in the pEGFP-N1 plasmid (CLONTECH, Palo Alto, CA). The corresponding control vector consisted of the signal peptide of TrkA fused in frame to enhanced GFP within pEGFP-N1. The CPE dominant negative construct or the control plasmid were transfected into INS-1 cells, a ß-cell line, and secretion experiments were carried out as described below.
Design of Short Interfering RNA (siRNA)
To deplete CPE in INS-1 cells, siRNAs were synthesized using the Silencer siRNA Construction Kit by Ambion (Austin, TX). Target sequences within the rat CPE mRNA sequence (38) were selected and a search with basic local alignment and search tool showed that 10 of the selected targets were not homologous with other known rat mRNAs. dsRNAs were synthesized against these 10 targets and transfected into INS-1 cells, which were then screened for the extent of CPE depletion by Western blot analysis of cell lysates. One set of dsRNA, spanning nucleotides 329349, significantly depleted CPE levels in INS-1 cells and was used in subsequent secretion experiments. In all screening and secretion experiments, 200 nM of dsRNA was transfected into six-well plates of INS-1 cells using Oligofectamine (Invitrogen) for 72 h.
Secretion Experiment in siRNA or Dominant Negative CPE Transfected INS-1 Cells
After transfection with either 100 µg of the dominant negative CPE construct or 200 nM of the dsRNA, INS-1 cells were preincubated for 3 h at 37 C in glucose-free DMEM supplemented with 1.6 mM glucose and 0.5 mg/ml BSA (basal media). Media were removed, and the cells were then incubated for an additional 3 h in either basal media or media containing 10 mM glucose, 0.5 mg/ml BSA, 1 µM phorbol 12-myristate-13-acetate, 1 µM 3-isobutyl-1-methylxanthine, and 1 µM tolbutamide as previously described (39). Sep-Pak extractions for media and cells were carried out as described previously (40), and ILI was measured in both cell extracts and media by RIA. Both basal and stimulated insulin release was expressed as the percent of total insulin-like immunoreactivity from both media and cells.
In Vitro Binding Experiments
Secretory granule membranes were prepared from bovine intermediate lobe pituitary glands as previously described (18). Recombinant WT proinsulin, and B10, R65L, and R65P proinsulin were prepared from a bacterial expression system as previously described (25). Ten micrograms of membrane protein were incubated with 450,000 cpm of [125I]insulin (at pH 5.5 and 6.5), or with 450,000 cpm of [125I] WT proinsulin, B10, R65L, or R65P proinsulin at pH 6.5, as described previously (9, 21). To determine specific binding to CPE, displacement of proinsulin/insulin binding was carried out in the presence of 100 µM N-POMC1-26, which contains the sorting signal of POMC that binds to CPE, as previously described (9, 21).
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ACKNOWLEDGMENTS
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We thank Professor Sir Tom Blundell (Cambridge University, Cambridge, UK) for helpful discussions on the insulin structure and sorting motif, Dr. S. Milgram (University of North Carolina, Chapel Hill, NC), for the anti-TGN38 antibody and Dr. P. Arvan (Albert Einstein College of Medicine, Bronx, NY), for the INS-1 cells. We also thank Drs. Niamh Cawley and C. Stratakis (National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD) and Drs. P. Lowenstein, and M. Castro (Cedars-Sinai Medical Center, Los Angeles, CA) for helpful suggestions and critical reading of the manuscript.
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FOOTNOTES
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S.D. is a recipient of a Canadian Diabetes Association Postdoctoral Fellowship.
S.D. and F.S.S. contributed equally to this work.
Present address for S.J.M.: Aegera Therapeutics, Montreal, Quebec, Canada H3E 1A8.
Present address for S.D.: Lawson Health Research Institute, London, Ontario, Canada N6A 4V2.
Abbreviations: ASLE, AB13 SB17 LA16 EA17; CPE, carboxypeptidase E; dsRNA, double-stranded RNA; ELSA, EB13 LB17 SA16 AA17; ER, endoplasmic reticulum; GFP, green fluorescent protein; ILI, insulin-like immunoreactivity; PDB, Protein Data Bank; POMC, proopiomelanocortin; RNAi, RNA interference; RSP, regulated secretory pathway; siRNA, short interfering RNA; TGN, trans-Golgi network; WT, wild-type.
Received for publication November 18, 2002.
Accepted for publication June 16, 2003.
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