Identification of a Novel Prohormone Sorting Signal-Binding Site on Carboxypeptidase E, a Regulated Secretory Pathway-Sorting Receptor

Chun-Fa Zhang, Christopher R. Snell and Y. Peng Loh

Section on Cellular Neurobiology (C.-F.Z., Y.P.L.) Laboratory of Developmental Neurobiology National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
Novartis Institute for Medical Research (C.R.S.) London, WCIE 6BN, United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10,Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg 259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peptide hormones and neuropeptides, unlike other secretory proteins that are secreted constitutively, are specifically packaged into granules of the regulated secretory pathway (RSP), uniquely present in neuroendocrine cells. They are released in a calcium-dependent manner upon stimulation. These molecules are synthesized as larger precursors (prohormones/proneuropeptides) at the endoplasmic reticulum (ER). They are then transported and sorted at the trans-Golgi network into secretory granules (1, 2) where they are processed to bioactive peptides. For many prohormones, sorting to the RSP secretory granules is a prerequisite for processing. However, much remains to be learned about the sorting mechanism. Our previous studies have provided evidence that the sorting of prohormones to the RSP at the trans-Golgi network occurs by a receptor-mediated mechanism (3, 4). A sorting signal motif that is sufficient and necessary for targeting the prohormone POMC to the RSP has been identified (5, 6, 7). This motif consists of two acidic residues (Asp10, Glu14) and two aliphatic hydrophobic residues (Leu11, Leu18) exposed on the surface of an amphipathic loop (residues 8–20) (Fig. 1Go). Molecular modeling of proenkephalin, using POMC as a template and the x-ray crystal structure of insulin, revealed a similar sorting signal motif in both these molecules (Fig. 1Go). As in POMC, two acidic residues and two aliphatic hydrophobic residues were exposed on the surface of a loop structure in the N terminus of proenkephalin and in the A and B chains of insulin. In addition, GH, which was found to be constitutively secreted from Cpefat/Cpefat mouse anterior pituitary (9), also has a similar motif, as revealed by the x-ray crystal structure (PDB ID: 1HGU; Brookhaven National Laboratories, Brookhaven, NY). However, in chromogranin A, which does not depend on carboxypeptidase E (CPE) for sorting to the regulated secretory pathway (8), such a motif was not evident. Analysis to date suggests that the POMC-sorting motif is present in other (pro)hormones that use CPE as a sorting receptor.



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Figure 1. Low-Energy Conformers for POMC1-24, Proenkephalin1-41, and the x-Ray Structure of Porcine Insulin (11 )

The sorting signal motif already described (6 ) for N-POMC corresponds to residues D10, L11, E14, and L18. The corresponding residues in the modeled proenkephalin structure (residues D18, I19, E29, L32) are shown, as are the corresponding residues in the x-ray structure of porcine insulin (LA16, EA17, EB13, and LB17). The {alpha}-carbon atoms of the proposed sorting motif overlay with an RMS fit of ~1 Å.

 
Using N-POMC1-26, which contains the POMC-sorting signal motif, as a ligand in binding studies, a sorting receptor was identified as membrane CPE (3, 7). Two other lines of evidence further indicate that CPE is a sorting receptor in vivo. First, depletion of CPE in Neuro 2a cells by antisense RNA resulted in the missorting of POMC (3), proenkephalin, and proinsulin (8) to the constitutive pathway. Second, Cpefat/Cpefat mice with severely depleted levels of a mutant CPE in the pituitary (9, 10) showed constitutive secretion of large amounts of POMC (9). In addition, these mutant mice did not show the normal meal-stimulated rise in circulating CCK, consistent with a role of CPE as a sorting receptor for packaging hormones into secretory granules of the RSP (11).

In the present study, molecular modeling of CPE was carried out to predict the RSP sorting signal-binding domain. Two basic residues on CPE to which the two acidic residues of the RSP-sorting signal motif on the prohormones could bind were identified. Mutagenesis of those two basic residues, expression of the mutant CPEs in Sf9 cells using the baculovirus system, and prohormone binding studies with Sf9 cell membranes containing mutant CPEs were carried out to test the model. The study identified residues Arg255 and Lys260 on CPE as the putative prohormone sorting signal-binding site, providing further evidence in support of specific prohormone-CPE interaction at the molecular level to mediate sorting of prohormones to the regulated secretory pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Putative Sorting Signal Motifs on Proenkephalin and Proinsulin
Molecular modeling of N-POMC1-26 has previously revealed a RSP-sorting signal motif on POMC consisting of residues Asp10, Leu11, Glu14, Leu18, exposed on the surface of an amphipathic loop between Cys8 and Cys20 (Fig. 1Go) (6). Deletion and site-directed mutagenesis to disrupt the amphipathic loop of N-POMC1-26 (5, 6) led to the missorting of POMC to the constitutive secretory pathway. Here we show by immunocytochemistry that transfection of POMC mutated at the four exposed residues, Asp10, Leu11, Glu14, Leu18, into Neuro 2a cells resulted in no punctate immunostaining in the neurites (Fig. 2CGo), consistent with lack of packaging of mutant POMC into dense core secretory granules. These cells revealed primarily perinuclear immunostaining of mutant POMC that overlapped with wheat germ agglutinin staining of the Golgi (Fig. 2DGo). This is in contrast to cells expressing wild-type POMC that showed punctate immunostaining along and at the tips of neurites (Fig. 2AGo), typical of hormone packaging into regulated dense-core secretory granules. Untransfected cells did not show any positive immunostaining (Fig. 2BGo). These results support our prediction that residues Asp10, Leu11, Glu14, Leu18 on N-POMC constitute the motif that is necessary for sorting POMC into the RSP.



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Figure 2. Immunolocalization of WT and Mutant POMC Transfected into Neuro 2a cells

A and B, Rhodamine staining for WT POMC in transfected (A) and untransfected Neuro 2a cells (B). Note the punctateness in the staining along the neurites (arrows). C, Rhodamine staining for mutant POMC in transfected Neuro 2a cells. D, FITC-wheat germ agglutinin staining of the Golgi (arrows) for the cells presented in panel C. Bar, 10 µm.

 
Molecular modeling of proenkephalin1-41 also revealed a sorting motif similar to POMC, consisting of two acidic and two hydrophobic residues: D18, I19, E29, and L32 (Fig. 1Go). Likewise, a similar motif was found on the surface of insulin as revealed by the x-ray structure (12) (Fig. 1Go). This motif on insulin is maintained in proinsulin as revealed by nuclear magnetic resonance studies (13). Thus, putative RSP-sorting signal motifs similar to POMC are present in other prohormones that could interact with CPE to facilitate sorting to the regulated secretory pathway.

Molecular Modeling of CPE
Molecular modeling of CPE was carried out to identify a possible binding site for the POMC-sorting signal. Membrane CPE contains a pre- (signal peptide) sequence followed by a proregion at the N terminus of the enzyme (Fig. 3AGo). The sorting signal-binding site and the C-terminal hydrophobic tail for membrane anchoring are shown in Fig. 3AGo. Since the CPE inhibitor, guanidinoethyl mercaptosuccinic acid, was previously shown not to inhibit N-POMC1-26 binding to bovine pituitary intermediate lobe secretory granule CPE (3), the modeling focused on domains that were unique to CPE, outside of the catalytic site. The family of carboxypeptidases A, B, T, M, N, and E were aligned using Pileup (Genetics Computing Group, Madison, WI) taking as reference points the conserved zinc-binding and catalytic residues. The location of the binding motif for the RSP-sorting signal was predicted to be unique to CPE, exposed on the protein surface, and have suitable side chains to bind to the acidic side chains (Asp10 and Glu14) in the RSP-sorting signal (N-POMC8-20). High-resolution x-ray crystallographic structures are available for carboxypeptidase A, B and T, of which the latter is the most homologous to CPE. Sequence alignment of CPE and CPT readily identified CPE254-273 as an inserted loop with the required structural properties. The structural analysis predicted this sequence to be hydrophilic, surface located, and to have two ß-turns at positions 255 and 261. The sequence CPE254-273 was modeled and manually docked to N-POMC as described in Materials and Methods to give the bound conformation shown in Fig. 3BGo (the two structures have been drawn apart slightly to better view the side chain complementarity between the signal motif and receptor-binding domain).



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Figure 3. Diagrammatic Representation and Model of CPE

A, Diagrammatic representation of the CPE molecule showing the pre- and proregion, the sorting signal-binding site and C-terminal hydrophobic membrane-binding tail (MBD). B, Model of N-POMC and CPE254-273 showing the complementarity of Arg255 (R255), Lys260 (K260) in CPE, and Asp10 (D10) and Glu14 (E14) in N-POMC.

 
Binding of N-POMC1-26 to Recombinant CPE
To test the above model of the prohormone sorting signal-binding site, in vitro binding assays using recombinant CPE were established. Full-length membrane CPE was expressed in Sf9 insect cells using the baculovirus expression system. Figure 4AGo2 shows a confocal micrograph of Sf9 cells infected with recombinant virus carrying full-length CPE and immunostained with an N-terminal CPE antibody previously described (3). Note that most of the staining is highly concentrated around the cells, indicating that the CPE is primarily localized to the plasma membrane. No immunostaining was found in cells infected with CPE using a preimmune antiserum (Fig. 4AGo1). Fig 4BGo shows a Western blot of CPE from Sf9 cell membranes. Cells infected with the AcNPV virus carrying the vector alone without CPE (lane 1), and uninfected cells (lane 2) showed no CPE expression, whereas cells expressing full-length CPE showed a correctly sized band of ~55 kDa that was stained with CPE antibodies directed against the proregion (lane 3). Similar results were obtained with antibodies directed against the N terminus of CPE (Fig. 8AGo, lanes 1–3). Very little CPE staining was observed in Western blots of the medium, indicating minimal secretion of membrane CPE (data not shown). This further supports the immunocytochemical data that the recombinant CPE expressed in Sf9 cells is primarily anchored to the plasma membrane and most, if not all, of it is in the proform.



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Figure 4. Immunolocalization and Western Blot of CPE

A1, FITC immunofluorescence of CPE in infected Sf9 cells stained with preimmune antiserum. A2, Confocal micrograph showing FITC immunofluorescence staining of CPE on the surface of CPE-infected Sf9 cells. Bar, 10 µm. B, Western blot of CPE in cell membranes from uninfected cells (lane 1), Sf9 cells infected with AcNPV alone (lane 2), and Sf9 cells infected with mouse CPE (lane 3). The CPE proregion antisera nos. 1 and 2 mixed 1:1 were used at 1:2000 dilution.

 


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Figure 8. [125I]N-POMC1-26 Binding to CPE

A, Western blot of CPE from membranes (20 µg protein) of Sf9 cells expressing WT and the various CPE mutations (M1–M9 shown in Fig. 7Go) using the CPH5–2 antiserum. Uninfected cell membranes (lane 1) and cells infected with AcNPV virus alone (lane 2) showed no CPE expression. WT CPE (lane 3) and mutant CPEs, M1–M9, in infected cells are shown (lanes 4–12, respectively). B, Counts per min [125I]N-POMC1-26 bound to WT and mutant CPEs on Sf9 cell membranes. The numbers under the bars correspond to the Western blot numbering described in panel A. Values shown are the mean ± SEM assayed in triplicate from one membrane preparation for each mutation. The nonspecific membrane binding (lanes 1 and 2) was not subtracted from the bars shown (lanes 3–12). Results shown are representative of a total of three different infections and membrane preparations for each mutant. C, The counts per min [125I]N-POMC1-26 bound shown in panel B was normalized for the amount of CPE present in each sample, determined in arbitrary units by scanning the Western blots shown in panel A. The normalized counts per min for WT CPE control was made equal to 100%, and normalized values of the negative controls (lanes 1 and 2) or mutants (lanes 4–12) were calculated as percent relative to wild type. The numbers under the bars correspond to the Western blot numbering described in panel A.

 
An intact cell-binding assay using N-POMC1-26, which contains the RSP-sorting signal, was carried out to determine whether the membrane CPE in Sf9 cells was exposed to the cell surface. Sf9 cells were fixed and then incubated with N-POMC1-26 followed by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodimide-HCl (EDC). The cells were then assayed by immunostaining with N-POMC1-26 antibodies. Figure 5Go shows the immunocytochemical localization of CPE on the plasma membrane of infected Sf9 cells expressing CPE (Fig. 5BGo), but not in uninfected cells (Fig. 5AGo). Uninfected cells incubated with N-POMC1-26 showed no N-POMC1-26 immunostaining on the cell surface (Fig. 5CGo1), in contrast to cells that were infected with CPE (Fig. 5DGo). Furthermore, Sf9 cells infected with CPE showed no immunostaining when incubated with a different POMC peptide, ß-endorphin1-31, which does not contain the RSP-sorting signal motif (Fig. 5CGo2). These results indicate that N-POMC1-26 bound specifically to CPE, which was anchored in the Sf9 cell membranes facing the outside of the cells after exocytosis. This is further substantiated by the observation that the use of Triton X-100, which renders the cells permeable to proteins, was not necessary for the binding of N-POMC1-26 to CPE to occur (data not shown).



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Figure 5. Immunocytochemical Localization of CPE on Uninfected (A) and Infected (B) Sf9 Cells

Note the FITC immunofluorescence on the plasma membrane of the CPE-infected cells. N-POMC1-26 was incubated with uninfected (C1) and CPE-infected cells (D); and ß-endorphin1-31 was incubated with CPE-infected cells (C2). The ligands on the cells were then cross-linked, followed by immunodetection using antibodies against these peptides. Note the N-POMC1-26 immunofluorescence on the surface of infected cells, indicating binding of N-POMC1-26 to CPE on the plasma membrane, but not uninfected cells. Note also the lack of staining of CPE-infected cells incubated with ß-endorphin1-31. Bar, 10 µm.

 
A cell membrane-binding assay was established to demonstrate specific binding of prohormones to recombinant CPE. Figure 6AGo shows the effect of increasing concentrations of N-POMC1-26 on [125I]N-POMC1-26 binding to recombinant membrane CPE. CPE bound [125I]N-POMC1-26 specifically in a N-POMC1-26-displaceable manner. However, ßh-endorphin1-31 did not displace the binding. Scatchard analysis showed a dissociation constant (Kd) of 9.2 µM (Fig. 6BGo). To further demonstrate binding specificity, mutant N-POMC1-26 in which the essential residues, Asp10, Leu11, Glu14, Leu18, in the sorting signal motif (Fig. 1Go) were mutated to Ala10, Ser11, Ala14, Ser18, was tested. Table 1Go shows that there was no binding of [125I] mutant N-POMC1-26 to CPE compared with N-POMC1-26.



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Figure 6. Displacement of [125I]N-POMC1-26

A, Dose-dependent displacement of [125I]N-POMC1-26 binding to CPE on infected Sf9 cell membranes by N-POMC1-26 (squares) or ß-endorphin1-31 (diamonds). Each data point represents n = 3 ± SEM. B, Scatchard plot of displacement of [125I]N-POMC1-26 binding to CPE on Sf9 cell membranes.

 

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Table 1. Binding of N-POMC to Recombinant CPE on Virus-Infected Sf9 Cell Membranes

 
Binding of N-POMC1-26 to CPE Mutants
A series of CPE mutants was made to test the model (Fig. 3BGo) predicting that residues Arg255 and Lys260 were essential for the binding of the prohormone-sorting signal. The nine mutants that were made are shown in Fig. 7Go. Figure 8AGo shows Western blot analysis of the relative levels of CPE in cell membranes from Sf9 cells expressing the wild-type CPE and the various mutants (M1–M9, lanes 4–12). The level of background binding of ligand to membranes from uninfected cells and cells infected with AcNPV virus alone is shown in lanes 1 and 2, respectively. Figure 8BGo shows the counts per min of [125I]N-POMC1-26 binding to the Sf9 cell membranes. While there was significant binding of [125I]N-POMC to wild-type (WT) CPE (lane 3), there was only background binding to CPE mutants with Arg255 (lanes 4 and 5) or Lys260 (lanes 6 and 7) substituted by Ala or Ser. Similarly, mutant CPEs with both Arg255 and Lys260 substituted by Ala or Ser (lanes 8 and 9) also showed only background [125I]N-POMC1-26 binding. The results indicate that both these basic residues are essential for N-POMC1-26 binding to CPE. To further demonstrate the highly specific nature of N-POMC1-26 binding to these two basic residues, another basic residue in the domain, Arg259, which is adjacent to Lys260, was mutated to Ala to determine whether the ligand could discriminate between the basic residues. This mutant CPE (lane 10) showed [125I]N-POMC1-26 binding similar to WT CPE, indicating that Arg259 was not involved in the interaction of N-POMC1-26 with CPE. Mutation of one of the CPE enzymatic substrate-binding sites Gln146 -Arg147 -Gln148 to Ala146 -Ala147 -Ala148 had no significant effect on [125I]N-POMC1-26 binding (lane 11). Also, since the Cpefat/Cpefat mouse carries a mutation in Ser202 Pro202 (9), this mutation was made to determine whether [125I]N-POMC1-26 can bind to this mutant form of CPE. Membranes from Sf9 cells expressing this mutant contained lesser amounts of mutant CPE relative to WT CPE (Fig. 8AGo, compare lanes 3 and 12), presumably due to degradation in the ER or in proteosomes after exit from the ER (14). As a result, there was reduced binding of [125I]N-POMC1-26 to CPE202 (Fig. 8BGo, lane 12). However, when the binding was corrected for the amount of mutant CPE202 present relative to WT control, the relative binding was similar to control (Fig. 8CGo, lane 12 vs. lane 3). The amount of all the other CPE mutants present on the Sf9 cell membranes was similar to WT CPE. Analysis of the relative binding of [125I]N-POMC1-26 correcting for the mutant CPE present (Fig. 8CGo), compared with the WT CPE, further confirmed that mutation of Arg255 and Lys260 (lanes 4–9) eliminated binding of [125I]N-POMC1-26 to CPE, but mutations to Arg259 (lane 10) or the substrate binding site (lane 11) had no effect.



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Figure 7. Partial Amino Acid Sequence of WT CPE Showing a Substrate-Binding Domain, the Cpefat/Cpefat Mouse Mutation (Ser202), and the Prohormone Sorting Signal-Binding Domain

The nine CPE mutations (M1-M9) and the residues substituted are shown. The amino acid letter codes are as follows: N, Asn; R, Arg; P, Pro; A, Ala; C, Cys; K, Lys; S, Ser.

 
Interestingly, no binding of [125I]N-POMC1-26 to soluble CPE was detected despite using 3 times the amount of CPE as was used for membrane-binding assays (data not shown), indicating that CPE must be anchored to membrane to bind N-POMC1-26.

Binding of Proinsulin and Proenkephalin to CPE and Mutants
Evidence that proinsulin and proenkephalin bind to the same receptor, CPE, as N-POMC1-26 is shown in Table 2AGo. [125I]N-POMC1-26 binding to recombinant membrane CPE was completely displaced by 10 µM proinsulin and proenkephalin. Figure 9CGo shows that the displacement of [125 I]N-POMC1-26 by proinsulin is competitive. Specific binding of proinsulin to CPE was further demonstrated by the immunostaining of proinsulin on the surface of intact Sf9 cells infected with CPE (Fig. 9BGo), but not on uninfected cells (Fig. 9AGo).


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Table 2. Binding of Prohormone to Recombinant CPE on Infected Sf9 Cell Membranes

 


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Figure 9. Displacement of [125I]N-POMC1-26 Binding to CPE

A and B, FITC immunofluorescence of proinsulin on the plasma membrane of cells infected with CPE (B) and uninfected cells (A). Proinsulin was incubated with uninfected and CPE-infected cells. The ligand on the cells was then cross-linked followed by immunodetection using an antibody that cross-reacts with proinsulin. Note the proinsulin immunofluorescence on the surface of infected cells, indicating binding of proinsulin to CPE on the plasma membrane, but not on uninfected cells. Bar, 10 µm. C, Dose-dependent displacement of [125I]N-POMC1-26 binding to CPE on infected Sf9 cell membranes by proinsulin. Each data point represents n = 3 ± SEM.

 
The binding of iodinated proenkephalin and proinsulin to WT CPE, mutant CPE202, and the mutant CPEs with substitutions in Arg255 and Lys260 for Ser is shown in Table 2BGo. [125I]proenkephalin and [125I]proinsulin bound to WT and mutant CPE202 in a similar manner. However, binding of proenkephalin and proinsulin to single (M2) (data not shown) or double Arg255 and Lys260 (M6) substituted CPE mutants (Table 2BGo) were poor, or none at all, indicating that these two residues are also necessary for the binding of the putative RSP-sorting signal of these two prohormones (Fig. 1Go), similar to N-POMC1-26.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular modeling studies predicted that a domain on CPE between Arg255 and Lys260 could be involved in binding the RSP-sorting signal motif of POMC. To test this model, a binding assay using recombinant full-length CPE or mutant CPEs expressed in Sf9 cells was used. [125I]N-POMC1-26 bound specifically to recombinant pro-CPE localized on the plasma membranes of Sf9 cells, and the binding was displaced by unlabeled N-POMC1-26 in a dose-dependent manner. The Kd of 9.2 µM was similar to the previously reported Kd (6 µM) for bovine pituitary intermediate lobe se-cretory granule membrane CPE (7). Thus, the N-POMC1-26 binding was unaffected by the presence of the 15-amino acid proregion present in the recombinant CPE. The specificity of the binding was fur-ther shown by the lack of binding of mutated N-POMC1-26, which has the four exposed residues (Asp10, Leu11, Glu14, Leu18) in the motif (see Fig. 1Go) substituted for Ala10, Ser11, Ala14, Ser18. This result further illustrates that these residues are essential for binding to the sorting receptor CPE. Molecular modeling studies suggest that there is ionic interaction between the acidic residues within the POMC RSP- sorting signal (Asp10, Glu14) with the two basic residues Arg255 and Lys260 in the CPE-binding domain. This is also consistent with the pH dependence of the binding of the RSP-sorting signal with CPE in vitro (3) and sorting to the RSP in vivo (15). Experimental evidence for this interaction was provided by the demonstration that when Arg255 and Lys260 on mouse CPE were substituted individually or together for Ala or Ser, binding of N-POMC1-26 to these CPE mutants expressed on Sf9 cell plasma membranes was eliminated. The precision of the ligand binding to these two basic residues was exemplified by the demonstration that substitution of Arg259, adjacent to Lys260, had no effect on the binding of N-POMC1-26. Furthermore, when residues Gln146-Arg147-Gln148 on CPE, of which Arg147 is involved in the binding of the COOH group of the basic residues of the substrate (16), were mutated, there was no significant effect on the binding of N-POMC1-26.

The ionic interaction involving Arg255 and Lys260 on CPE appears not to be limited to the POMC RSP-sorting signal, since mutation of these two basic residues on CPE also eliminated binding of proinsulin and proenkephalin. Both these prohormones have predicted RSP-sorting signal motifs with two acidic and two aliphatic hydrophobic residues in a configuration similar to Asp10, Leu11, Glu14, Leu18 in POMC (Fig. 1Go). Therefore, the Arg255 to Lys260 domain on the sorting receptor CPE, which is highly conserved among all species where the sequence is known, appears to be a common binding site for several putative prohormone RSP-sorting signals.

The results of this study, showing highly specific binding between prohormone and CPE in a saturable manner, are consistent with a possible role of membrane CPE as a RSP-sorting receptor for a number of prohormones. In addition, we have also shown that membrane CPE, which is enzymatically poorly active (17), is tightly bound to secretory granule membranes in a Triton X-100/1 M NaCl-insoluble manner (7), characteristic of a membrane-bound receptor. The binding domain in CPE for the sorting signal of POMC is independent of the enzymatic substrate site and available only when CPE is bound to the membrane. This further segregates the initial role of membrane CPE as a sorting receptor in the secretory pathway vs. its subsequent role as a soluble enzymatically active exopeptidase in the secretory granule in removing C-terminal basic residues from cleaved peptide hormones (17, 18).

The importance of CPE in sorting of prohormones in vivo was demonstrated by studies in which depletion of CPE in Neuro 2a cells by antisense CPE RNA resulted in missorting of POMC (3), proenkephalin, and proinsulin (8) to the constitutive pathway. In addition, we showed that Cpefat/Cpefat mice depleted of mutant CPE exhibited constitutive secretion of large amounts of POMC from intermediate and anterior lobes of their pituitary glands (3, 9). While recent studies with NIT 2 and NIT 3 cells, immortalized pancreatic cell lines from Cpefat/Cpefat mice, have shown regulated secretion of immunoreactive insulin, it was significantly less than that from immortalized pancreatic NIT 1 cells from control mice (19). Interestingly, there was as much mutant CPE202 in the NIT 3 cells as WT CPE in NIT 1 cells. Given that our present study has shown that CPE202 can exit the ER in Sf9 cells and bind proenkephalin and proinsulin as efficiently as WT CPE, it is possible that some CPE202 in the NIT 2 and NIT 3 cells may have exited the ER and are able to serve as sorting receptors. Similarly, in another study carried out by Irminger et al. (20) using Cpefat/Cpefat mice, regulated secretion of proinsulin was also observed in pancreatic islet cells. The design of this release study precludes detection of any constitutive secretion of proinsulin that may have occurred during the stimulation period, since no parallel experiments tracking unstimulated release of proinsulin for the same period was carried out. Therefore the amount of stimulated secretion of proinsulin in the Cpefat/Cpefat mice may, in fact, have been less than that reported. In addition, like the NIT3 cells, significant levels of immunoreactive CPE202, including the mature form, have been detected by Naggert et al. (10) in the pancreatic islet cells of Cpefat/Cpefat mice, although pituitary had virtually none. Presence of the mature form in the islets suggests that CPE202 exited the ER, since maturation has been shown to occur in the trans-Golgi network (21). In the context of the present study, it is therefore possible that there are sufficient levels of immunoreactive CPE202 in the pancreatic islets of the Cpefat/Cpefat mice to bind and sort proinsulin to the RSP. Since CPE levels were not determined in the islets of the mice used in the study of Irminger et al. (20), the possibility exists that significant amounts of CPE202 in the islets may account for their results. Nevertheless, a different mechanism for sorting proinsulin to the RSP in the islets, which does not exist in Neuro 2a cells, cannot be ruled out. Further studies will clarify the role of CPE as a sorting receptor in different cell types and for different prohormones.

In summary, molecular modeling and site-directed mutagenesis studies have identified a putative motif between residues 255 and 260 on CPE that can bind the prohormone RSP-sorting signal. Within this domain, two basic residues, Arg255 and Lys260, appear to be essential for the ionic binding of the acidic residues, Asp10 and Glu14, within the POMC RSP-sorting signal, as well as proenkephalin and proinsulin, which also have predicted sorting signal motifs that contain two acidic residues. RSP-sorting signal domains have been identified for other RSP proteins, e.g. chromogranin B (22, 23), the processing enzymes PC5A (24), PC2 (25), and peptidylglycine {alpha}-amidating monooxygenase (26). The mechanism by which these sorting signal domains serve to target these proteins into the regulated secretory pathway has not been elucidated. Clearly, CPE does not act as a sorting receptor for chromogranin A (7, 8) and hence other sorting receptors and mechanisms may exist (27). However, the understanding of how the POMC sorting signal and the proenkephalin and proinsulin putative sorting signals can specifically interact with CPE, as elucidated from this study, is consistent with the model of a receptor-mediated mechanism for sorting prohormones to the RSP.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Full-length mouse CPE cDNA was cloned from mouse Lambda ZAP II library (Stratagene, La Jolla, CA) screening with a CPE cDNA sequence probe. Deep Vent DNA polymerase, T4 DNA ligase, and restriction endonucleases were from New England Biolabs (Beverly, MA). Insect Sf9 cell line, pAcGP67 expression vector, and linearized BaculoGold DNA were from Pharmingen (San Diego, CA). N-POMC1-26 was from Phoenix Pharmaceuticals, Inc. (Mountain View, CA), and mutated N-POMC1-26 (Asp10, Leu11, Glu14, Leu18, to Ala10, Ser11, Ala14, Ser18) was custom synthesized by Peninsula Laboratories (Belmont, CA). ßh-Endorphin1-31 was from Peninsula Laboratories (Belmont, CA). N-POMC1-26 and mutant N-POMC1-26 were iodinated by Covance Inc. (Vienna, VA.) using 125I-labeled Bolton-Hunter Reagent (Amersham, Arlington Heights, IL). Proenkephalin and proinsulin (gifts from I. Lindberg and K. Vad, respectively) were iodinated by Covance, Inc., using the Chloramine T procedure. The rabbit antiserum raised against N-POMC1-26 was a gift from Phoenix Pharmaceuticals, Inc.; the rabbit antiserum DP3 was raised against human ß-endorphin1-31by Covance Inc. The insulin antiserum was purchased from Peninsula Laboratories. The antisera (nos. 1 and 2) against the proregion of CPE was a gift from L. Fricker. EDC and N-hydroxysuccinimide were from Pierce Immunologicals (Rockford, IL). All other chemicals were of analytical grade.

Immunocytochemistry of Neuro 2a Cells Expressing WT and Mutant POMCs
Mutant POMC with D10L11E14L18 substituted for A10,S11A14S18 was generated by PCR using bovine POMC cDNA as a template and a mutagenic oligonucleotide as primer. WT and mutant POMC cDNA fragments were subcloned into the mammalian expression vector pcDNA3.1 (from Invitrogen, Carlsbad, CA) at the restriction sites, NheI and EcoRV. The two constructs were transformed into DH5{alpha} bacteria and plasmid DNAs were prepared. The mutant POMC cDNA was confirmed by sequencing. Neuro 2a cells, a neuroendocrine cell line, were transfected with WT and mutant POMC cDNA using lipofectin (Life Technologies Inc. Gaithersburg, MD). After 48 h, the cells were fixed, permeabilized, blocked with 10% goat serum in PBS, incubated with antibody against ACTH (DP4, which also cross-reacts with POMC, 1:5000 dilution), and then stained with goat antirabbit serum conjugated with rhodamine (Boehringer Mannheim, Indianapolis, IN.) (6). Wheat germ agglutinin conjugated with fluorescein isothiocyanate (FITC, EY Laboratories, Inc., Mateo, CA, 1:250 dilution) was used to stain the Golgi apparatus. Images were captured using a TEC-470 charge-couple device color camera on a Nikon Optiphot epifluorescent microscope (Nikon, Inc., Melville, NY) coupled to a Macintosh computer using Adobe Photoshop software (Adobe Systems, Mountain View, CA).

Baculovirus Expression of WT and Mutant CPEs
Ten cDNA fragments encoding mouse Pro-CPE or mouse mutant Pro-CPE (M1: R255 to A; M2: R255 to S; M3: K260 to A, M4: K260 to S; M5: R255 and K260 to A; M6: R255 and K260 to S; M7: R259 to A; M8: NRN146–148 to AAA and M9: S202 to P) generated by PCR were inserted into the pAcGP67 baculovirus expression vector. These ten constructs were then transformed into DH5{alpha} bacteria (Life Technologies), and the plasmid DNAs were prepared using Qiagen Maxiprep Kits (Qiagen, Valencia, CA) and sequenced with PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA). WT and mutant Pro-CPE plasmid DNAs were cotransfected with linearized BaculoGold AcNPV DNA (Pharmingen) into Sf9 cells, using the calcium phosphate method. The transfected cells were maintained at 27 C in TMN-FH medium containing 10% FBS, 50 IU/ml penicillin-streptomycin, and 0.125 µg/ml Fungizone for 5–6 days. The recombinant viruses were purified and identified by plaque assay. For Pro-CPE or mutant Pro-CPE expression, Sf9 cells were infected with recombinant baculoviruses at a multiplicity of infection of 5. The medium and the cells were harvested 2.5 days post infection.

Preparation of Sf9 Cell Membranes
Infected and uninfected Sf9 cells were harvested, washed with PBS, and homogenized in a tissue grinder (Potter-Elvehjem), with 50 strokes at 1800 rpm. The cell membranes were harvested by centrifugation at 100,000 x g for 45 min, and the pellet was suspended in binding buffer (50 mM MES buffer, pH 5.5, containing 120 mM NaCl and 5 mM KCl).

Membrane Binding Assay
The binding assay was carried out in binding buffer using 20 µg membrane protein in each assay except for Fig. 6Go where 10 µg protein were used. The total volume of the assay was 400 µl, and each assay was done in triplicate. [125I]N-POMC1-26 was added to the binding assay to a final concentration of 2 pM (~150,000 cpm), except for experiments in Fig. 8Go, where ~450,000 cpm were used. The binding reaction was performed at 4 C overnight. Bound N-POMC1-26 was then precipitated by the addition of 200 µl 0.5% {gamma}-globulin and 500 µl 24% polyethelene glycol (PEG 6000) to the reaction, followed by incubation on ice for 15 min and centrifugation at 3000 rpm for 30 min at 4 C. The supernatant was removed and the pellet counted on a Beckman {gamma}-counter. A blank without membranes was used for background subtraction. Specific N-POMC1-26 binding was calculated by subtracting the binding measured in uninfected, or AcNPV (virus without CPE)-infected Sf9 cell membranes. For the dose-dependent displacement binding studies, the extent of binding of [125I]N-POMC1-26 was determined in the presence or absence of various concentrations of unlabeled N-POMC1-26, or proinsulin. The binding studies with [125I] proenkephalin and [125I]proinsulin were as described for N-POMC1-26, except that the incubations were done at pH 6.5, the optimal pH for binding of these prohormones, and ~450,000 cpm of ligand were used.

Intact Cell Binding Assay
Sf9 cells were seeded in a four-well slide. At a cell density of 80% confluence, the cells were infected with recombinant baculovirus; 2.5 days post infection, the infected cells and uninfected cells were fixed with 3% HCHO for 1 h at 22 C. After two washes with PBS, the cells were blocked with 10% goat serum for 30 min at 22 C. They were then incubated with the ligand, N-POMC1-26, ßh-endorphin, or proinsulin (final concentration 100 µM) in binding buffer at 4 C overnight. The ligand was then cross-linked by adding EDC (final concentration, 1 mM) and N-hydroxysuccinimide (final concentration, 5 mM) to the cells at 22 C for 20 min. The cells were then washed three times with PBS, reacted with antiserum against N-POMC1-26 (1:500 dilution), ßh-endorphin (1:2000 dilution), or proinsulin (1:200 dilution) at 22 C for 2 h, followed by incubation with a second antibody coupled to FITC (1:1000) for 1 h at 22 C. After three washes with PBS, the cells were observed and images captured using a TEC-470 charge couple device color camera (Optronics Engineering, Goleta, CA) on a Nikon Optiphot epifluorescent microscope coupled to a Macintosh 8100/100 Power PC computer, using Adobe Photoshop 4.0 software.

Immunocytochemistry of CPE in Sf9 Cells
Sf9 cells were grown, infected, fixed, and blocked with goat serum as described above. The cells were then washed three times with PBS, reacted with CPE antiserum (CPH5–2, 1:500 dilution) raised against the N terminus of CPE, at 22 C for 2 h, followed by incubation with a second antibody coupled to FITC (1:1000) for 1 h at 22 C. After three washes with PBS, the cells were observed and images captured as described above or on a confocal microscope.

Western Blot Analysis of CPE
Samples were separated on 12% SDS PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was probed with CPE antiserum (CPH5–2), or pro-CPE antisera, and CPE was detected using the alkaline phosphatase method (picoBlue immunoscreening kit, Stratagene).

Molecular Modeling of CPE-Binding Motif and Complex with N-POMC
Sequence analysis of CPE and related carboxypeptidases was performed using the GCG suite of programs (Genetics Computing Group). The sequence (CPE254-273) was modeled with the C and N termini constrained to 5 Å as required if the loop were to be inserted in the CPT structure (a manuscript by C. R. Snell and Y. P. Loh detailing the construction of CPE by homology with CPT is in preparation). Low-energy conformers were generated using a combination of molecular dynamics and mechanics (Discover, MSI Inc., San Diego, CA). Conformers were sampled every picosecond during the molecular dynamics trajectory of 250 psec (CVFF forcefield, 600 K), and subject to extensive energy minimization using the Newton-Raphson algorithm until the gradient was less than 0.01 kcal Å-2. The lowest energy conformer was manually docked with POMC in the conformation previously reported aligning the Arg255 and Lys260 with Asp10 and Glu14 in POMC. Molecular mechanics were used to optimize the interaction between the two structures as described above for CPE254-273.

Molecular Modeling of N-POMC1-26 and Proenkphalin1-41
Molecular mechanics and molecular dynamics were performed using Insight II and Discover (Molecular Simulations, Inc., San Diego, CA). Each peptide sequence was assembled and disulfide bridges formed according to the known disulfide pattern (28). Gentle molecular dynamics at 300 K was performed for 25 psec, until the disulfide bond lengths had optimized. These structures were then minimized using Steepest Descents and a Newton-Raphson minimizer until convergence. These structures were then subjected to simulated annealing using molecular dynamics for 250 psec at 600 K, and molecular mechanics to minimize structures, sampled every picosecond from the dynamics trajectory, to convergence with gradient below 0.005 kcal Å-2. This gave 250 low-energy structures for each peptide, and from these, the lowest energy conformers were used for further structural studies.


    ACKNOWLEDGMENTS
 
We thank Drs. K. Vad (Novo Nordisk, Bagsvaerd, Denmark), I. Lindberg (Louisiana State University, Baton Rouge, LA), and L. Fricker (Albert Einstein College of Medicine, Yeshiva University) for gifts of swine proinsulin, rat proenkephalin, and proCPE antibodies, respectively. We thank Dr. J. Bonifacino for critical reading of this manuscript and Ms. Winnie Tam for performing the immunocytochemistry on Neuro 2a cells transfected with WT and mutant POMCs.


    FOOTNOTES
 
Address requests for reprints to: Dr. Y. Peng Loh, National Institutes of Health, Building 49, Room 5A38, Bethesda, Maryland 20892. E-mail:ypl{at}codon.nih.gov

Received for publication September 4, 1998. Revision received January 4, 1999. Accepted for publication January 5, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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