©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Addition of an Endoplasmic Reticulum Retention/Retrieval Signal Does Not Block Maturation of Enzymatically Active Peptidylglycine -Amidating Monooxygenase (*)

Hye-Young Yun , Betty A. Eipper (§)

From the (1)Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Peptidylglycine -amidating monooxygenase (PAM) catalyzes the COOH-terminal -amidation of neural and endocrine peptides via a two-step reaction carried out in sequence by the monooxygenase and lyase domains contained in this bifunctional protein. Peptide -amidation is thought to take place primarily in the secretory granules in which mature bioactive peptides are stored, and it is not known where in the secretory compartment newly synthesized PAM protein becomes enzymatically active. To address this question, PAM-3, a soluble bifunctional protein, was modified by addition of the KDEL endoplasmic reticulum (ER) retention/retrieval signal to its COOH terminus. PAM-3-KDEL protein stably expressed in hEK-293 cells or in AtT-20 cells was efficiently retained in the ER based on immunocytochemistry, pulse-chase experiments, and maintained endoglycosidase H sensitivity. The effect of the KDEL sequence was specific since PAM-3 with an inactive ER retention/retrieval signal (PAM-3-KDEV) moved through the secretory pathway like wild type PAM-3. In AtT-20 cells, PAM-3-KDEL was not subjected to the COOH-terminal endoproteolytic cleavage that generates a 75-kDa PAM protein from PAM-3 and PAM-3-KDEV. PAM-3-KDEL protein exhibited both monooxygenase and lyase activities with specific activities similar to those of the wild type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM catalyzes a reaction that occurs primarily in the secretory granules, newly synthesized PAM protein becomes enzymatically competent in the ER.


INTRODUCTION

Biosynthetic maturation of bioactive peptides in the neural and endocrine system involves a variety of post-translational processing steps during transit of precursor proteins through the secretory compartments(1, 2) . Endoproteolytic cleavage and removal of COOH-terminal basic amino acids take place initially in the trans-Golgi network (TGN),()continue in secretory granules and often result in the production of COOH-terminal Gly-extended peptides that are subsequently -amidated to produce active peptides. Peptide -amidation is often rate-limiting and essential for generating bioactive peptides(3, 4) .

Peptide -amidation is a two-step reaction catalyzed in sequence by monooxygenase and lyase domains contained in a bifunctional enzyme, peptidylglycine -amidating monooxygenase (PAM; EC 1.14.17.3). The first step of the reaction is catalyzed by peptidylglycine -hydroxylating monooxygenase (PHM) and requires copper, ascorbate, and molecular oxygen; the second step is catalyzed by peptidyl--hydroxyglycine -amidating lyase (PAL) and generates -amidated peptide and glyoxylate(5, 6, 7, 8, 9) . This modification is a late step in the maturation of bioactive peptides beginning in the TGN after endo- and exoproteolytic cleavages but occurring mostly in secretory granules(3, 10) .

Post-translational modifications are highly compartmentalized events, particularly in the secretory pathway. The compartments in which specific processing events occur are determined by the maturation/activation of the enzymes, restricted localization of the enzymes, availability of substrate, and the physicochemical milieu of the compartments such as pH, divalent cations, and cofactors(11, 12) .

In general, endo- and exoproteolytic cleavages of peptide precursors must precede peptide -amidation. The subtilisin-like endoproteases are involved in processing of prohormones. Furin, PC1, and PC2 undergo an autocatalytic cleavage that removes the pro-region and is required for production of fully active enzyme(13, 14, 15, 16) . Pro-region cleavage of furin and PC1 takes place in the endoplasmic reticulum (ER)(13, 17, 18, 19, 20) . Nevertheless, generation of catalytically active furin requires a post-Golgi event(21, 22) .

In the case of peptide -amidation, there is evidence suggesting the importance of both the targeting of PAM and compartment physicochemical milieu in establishing the restricted location of peptide -amidation. First, PAM protein is targeted to the secretory granules when expressed in AtT-20 corticotrope tumor cells(23) , and PAM proteins are enriched in dense core vesicles in the central nervous system(24) . Second, proopiomelanocortin-derived -amidated joining peptide immunoreactivity is detected in the TGN and secretory granules (10). Third, the TGN and secretory granules are the compartments in which Gly-extended peptide substrates and optimum pH are available.

Although PAM proteins are subjected to glycosylation, sialylation, sulfation, and endoproteolytic processing(23, 25, 26, 27) , little is known about where in the secretory compartment newly synthesized PAM proteins become enzymatically active. To address this question, we attached the ER retention/retrieval signal, KDEL, or an inactive homologue, KDEV (28), to the COOH terminus of a bifunctional soluble PAM protein (PAM-3) (Fig. 1). Both proteins were examined in stably transfected non-neuroendocrine cells (hEK-293) and neuroendocrine cells (AtT-20). We found that the PAM-3-KDEL protein was efficiently retained in the ER in both cell types and that ER-retained PAM-3-KDEL protein had enzyme activities similar to those of wild type PAM-3 and PAM-3-KDEV. Thus, acquisition of enzyme activities by PAM does not require transit beyond the cis-Golgi network.


Figure 1: Mutant PAM-3 proteins. A schematic diagram of mutant PAM-3 proteins with COOH-terminal ER retention signal is shown. PAM-3 is a splicing variant of the longest form of PAM (PAM-1) lacking both the non-catalytic region between PHM and PAL and the transmembrane domain between PAL and the COOH-terminal domain (CD). Irregular closed curve, N-linked oligosaccharide on Asn (25); arrowhead with vertical tic mark, paired basic amino acid endoproteolytic cleavage sites that may be used in AtT-20 cells (23). Tyr of PAM-3 is sulfated (26). All sites are numbered as in PAM-1, which is 976 residues in length.




MATERIALS AND METHODS

Plasmid Construction

pBluescript plasmids encoding PAM-3 with COOH-terminal KDEL or KDEV sequences were created using the polymerase chain reaction; pBS.KrPAM-3 was amplified using a sense primer in the PAL domain (nucleotides 2517-2533) and an antisense primer with an XbaI site (boldface) and codons for KDEL or KDEV following the COOH-terminal end of PAM-3 (underlined): 5`-CCTCTA-GACTACAGCTCGTCCTTGGAGGAAGGTGCAGGCTT-3` for KDEL and 5`-CCTCTAGACTACACCTCGTCCTTGGAGGAAGGTGCAGGCTT-3` for KDEV. The amplified fragment obtained was digested with XbaI and AatII (nucleotide 2620) and inserted into pBS.KrPAM-3 (23) from which the XbaI/AatII fragment had been removed, creating pBS.KrPAM-3-KDEL and pBS.KrPAM-3-KDEV. The cDNA region derived from polymerase chain reaction was verified by sequencing. The KrPAM-3-KDEL and KrPAM-3-KDEV cDNA was inserted into the pCIS.2CXXNH expression vector by ligation of the Bsp106I/XbaI fragment of pBS.KrPAM-3-KDEL or pBS.KrPAM-3-KDEV and the Bsp106I/XbaI vector fragment of pCIS.KrPAM-3.

Cell Culture and Transfection

hEK-293 cell lines were maintained in DMEM/F-12 (Life Technologies, Inc.) containing 10% fetal clone serum (Hyclone, Logan, UT) and antibiotics at 37 °C in 5% CO as described(23) . AtT-20 cells were maintained in the same medium supplemented with 10% NuSerum (Collaborative Research, Bedford, MA). Cells were passaged weekly. pCIS.KrPAM-3-KDEL and pCIS.KrPAM-3-KDEV plasmids were transfected into hEK-293 cells and AtT-20 cells using Lipofectin (Life Technologies, Inc.) as described (23). Transfected cells were grown in DMEM/F-12 containing antibiotics and 0.5 mg/ml G418 supplemented with 10% fetal clone serum (for hEK-293 cells) or 10% fetal clone serum and 10% NuSerum (for AtT-20 cells). G418-resistant cells were screened for expression of PAM proteins by immunocytochemical staining using PHM antibody or PAL antibody as described(29) .

PHM and PAL activities were measured as described (5, 6) using 0.5 µM -N-acetyl-Tyr-Val-Gly and 0.5 µM -N-acetyl-Tyr-Val--hydroxyglycine. Western blot analyses were carried out as described(30) . PAM proteins were detected using ECL (Amersham Corp.) with rabbit polyclonal antisera against the PHM domain (antibody 475; Ref. 25) and mouse monoclonal antibody against the COOH-terminal domain of PAM (6E6).()

Biosynthetic Labeling and Immunoprecipitation

Stably transfected cells were plated on 12-mm culture dishes coated with 25 µg/ml fibronectin (for hEK-293 cells) or 0.1 mg/ml poly-L-lysine (for AtT-20 cells) and grown a minimum of 36 h and were 50-75% confluent before experiments were begun. Biosynthetic labeling with [S]cysteine/methionine (Amersham Corp.) (0.3 mCi/well, 1-2 µM methionine) was performed as described (31). Cells were labeled for 15 min (pulse) and rinsed once with complete serum-free medium. Cells were then either extracted with 20 mM TES (pH 7.4), 10 mM mannitol, 1% Triton X-100 containing protease inhibitors as described (23) or incubated for varying periods of time in 300 µl of complete serum-free medium (chase). Immunoprecipitation of cell extracts and media was carried out as described using rabbit antibody to recombinant COOH-terminal domain of PAM (antibody 571)(25, 31) . Immunoprecipitated proteins were fractionated by SDS-PAGE on 10% acrylamide (0.27% N,N`-methylenebisacrylamide) gels and visualized by fluorography. The apparent molecular masses of the immunoprecipitated proteins were determined by comparison with prestained molecular weight standards (Rainbow standards; Amersham Corp.). Densitometric analyses were performed using an Abaton Scan 300/GS linked to an Apple Macintosh IIci and NIH Image 1.35 software (National Institute of Mental Health). N-glycanase and endoglycosidase H treatment of immunoprecipitated PAM proteins were performed as described(27, 32) .


RESULTS

hEK-293 cells and AtT-20 cells were stably transfected with expression vectors encoding PAM-3-KDEL or PAM-3-KDEV. We chose KDEV as a control sequence since it was inactive in ER retention and had no effect on normal secretion and processing when attached to the COOH terminus of proneuropeptide Y(28) . To examine the effect of the COOH-terminal KDEL sequence on the intracellular distribution of PAM-3, transfected cells were fixed, permeabilized, and visualized with a PAM antibody. Both hEK-293 and AtT-20 cells expressing PAM-3-KDEL showed a typical ER-staining pattern with a diffuse, reticular network-like pattern extending throughout the cytoplasm (Fig. 2, A and E). In contrast, hEK-293 and AtT-20 cells expressing PAM-3-KDEV protein exhibited staining patterns similar to those of wild type PAM-3 in the same cell type (Fig. 2, B, C and F, G)(23, 29) . PAM-3-KDEV expressed in hEK-293 cells exhibited concentrated staining in the perinuclear region, overlapping but more diffuse than a Golgi marker, wheat germ hemagglutinin. In transfected AtT-20 cells, PAM-3-KDEV displayed punctate staining throughout the cell body with concentrated staining at the tips of processes and some staining in the perinuclear region.


Figure 2: Immunofluorescence staining. hEK-293 cells (A-D) and AtT-20 cells (E-H) expressing PAM-3-KDEL (A and E), PAM-3-KDEV (B and F), PAM-3 (C and G), or no exogenous PAM (D and H) were fixed, permeabilized, and stained with rabbit polyclonal antibody to PAL (antibody 471; 1:1500; A, B, E, and F) or to PHM (antibody 475; 1:1500; C, D, G, and H) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:500; CalTag, San Francisco, CA). Scalebar, 20 µm.



To further investigate the effect of addition of the KDEL sequence on the trafficking of PAM-3, hEK-293 cells expressing PAM-3-KDEL or PAM-3-KDEV were incubated with [S]cysteine/methionine for 15 min (pulse) and then incubated with medium containing non-radioactive cysteine (200 µM) and methionine (114 µM) for 2 or 18 h (chase). Cell extracts and chase media collected from each time point were analyzed by immunoprecipitation and SDS-PAGE (Fig. 3A). After the 15-min pulse, 0.5-1% of the protein synthesized (trichloroacetic acid-precipitable radioactivity) could be identified as a 95-kDa PAM-3 protein using antiserum to the PAM COOH-terminal domain. After 2 or 18 h of chase, the newly synthesized PAM-3-KDEL protein was still retained in the cells without significant loss of protein. Very little PAM-3-KDEL protein (<2% of the newly synthesized PAM-3-KDEL) appeared in the chase medium even after an 18-h chase. In contrast, about 50% of the newly synthesized PAM-3-KDEV protein was secreted during the initial 2-h chase. This rate of secretion is similar to the observed rate of constitutive secretion of wild type PAM-3 from transfected hEK-293 cells (30-50% of PAM-3 in the cell/h)(26, 29) . After 18 h of chase, all of the PAM-3-KDEV found in the cell extract after the pulse was recovered in the chase medium with little loss of protein. Thus, the effect of the KDEL sequence on retention of PAM-3 in the cells is specific and efficient.


Figure 3: Pulse-chase experiment and endoglycosidase H treatment. A, three identical wells of hEK-293 cells expressing PAM-3-KDEL or PAM-3-KDEV were incubated with [S]cysteine/methionine for 15 min and harvested (0 h cell) or chased for the indicated times in complete serum-free medium. Equal amounts of cell extracts and chase media were immunoprecipitated with an antibody to the COOH-terminal domain of PAM (antibody 571) and analyzed by SDS-PAGE and fluorography. B, selected samples from this and another pulse-chase experiment were immunoprecipitated and subjected to Endo H or N-glycanase (N-gly) treatment; control samples (Con) were from the same incubations without enzymes. Samples were analyzed by SDS-PAGE and fluorography. Similar results were obtained in two independent studies.



PAM-3 contains a single N-linked oligosaccharide at Asn in the PAL domain (25) (Fig. 1). To test how far the cell-associated PAM-3-KDEL protein progressed through the cell, PAM-3 proteins immunoprecipitated from selected samples of cell extract or medium (Fig. 3A) were digested with endoglycosidase H (Endo H) or N-glycanase (Fig. 3B); acquisition of resistance to digestion with Endo H is diagnostic of transit from the ER to the medial-Golgi compartment(33) . Immediately after the pulse (0 h cell), both newly synthesized PAM-3-KDEL and KDEV proteins were Endo H-sensitive. PAM-3-KDEL remained Endo H-sensitive up to 18 h after its biosynthesis. Thus PAM-3-KDEL protein expressed in hEK-293 cells is efficiently retained in the ER without significant degradation for up to 18 h. In contrast, as expected from its similarity to wild type PAM-3, 50% of the newly synthesized PAM-3-KDEV protein was Endo H-resistant after a 1.5-h chase. The behavior of PAM-3-KDEV is consistent with its expected similarity to PAM-3, which has a t for ER to Golgi transit of about 1 h in AtT-20 cells(32) . PAM-3-KDEV protein that had been secreted into the medium was completely Endo H-resistant. These results together with immunocytochemistry data clearly demonstrate that PAM-3-KDEL protein is efficiently retained in the ER.

In transfected AtT-20 cells, which possess a regulated secretory pathway and secretory granules, PAM-3 protein (95 kDa) is targeted to the secretory granules and cleaved into a 75-kDa bifunctional PAM protein lacking its COOH-terminal domain (Fig. 1)(23) . This endoproteolytic cleavage is blocked upon incubation at 20 °C and is thought to occur after exit from the TGN, presumably in immature secretory granules(18) . Extracts of AtT-20 cells expressing PAM-3-KDEL or PAM-3-KDEV were analyzed by SDS-PAGE and immunoblot with PHM antibody (Fig. 4). Extracts from PAM-3-KDEL cells showed only a 95-kDa PAM-3-KDEL protein; no 75-kDa cleavage product was detected, indicating that PAM-3-KDEL protein failed to reach the site where the cleavage normally occurs. Extracts of PAM-3-KDEV cells contained similar amounts of 95-kDa PAM-3-KDEV and 75-kDa PAM derived from cleavage of PAM-3-KDEV. Thus the presence of the COOH-terminal KDEL signal specifically blocked targeting of PAM-3 to the secretory granules.


Figure 4: PAM-3-KDEL protein expressed in AtT-20 cells. Extracts of AtT-20 cells expressing PAM-3-KDEL or PAM-3-KDEV were assayed for PHM activity, and equal amounts of PHM activity (10, 5, and 2.5 nmol/h) from each extract were analyzed by immunoblot with PHM antibody (antibody 474) and ECL. The PHM specific activities of PAM-3-KDEL and PAM-3-KDEV cell extracts were 1100 to 1300 and 50 to 100 pmol/µg/h, respectively. Similar data were observed on two separate set of extracts.



Extracts of PAM-3-KDEL and PAM-3-KDEV AtT-20 cells were assayed for PHM activity. Western blot analysis indicated that samples with approximately equal amounts of PHM activity contained similar amounts of PAM protein (Fig. 4; densitometric values differed by <50% for PAM-3-KDEL and PAM-3-KDEV, n = 8). Since the endoproteolytic cleavage that separates the COOH-terminal domain from PAL affects PHM activity(34) , a more detailed analysis of the effect of the KDEL/KDEV sequence on enzymatic activity was carried out in hEK-293 cells, where endoproteolytic cleavages of this type do not occur.

hEK-293 cells expressing wild type PAM-3, PAM-3-KDEL, or PAM-3-KDEV were assayed for PHM and PAL activities. To compare the specific activities of wild type and mutant PAM-3 proteins, equal amounts of PHM activity (1 nmol/h) were subjected to immunoblot analysis (Fig. 5A). The amounts of wild type and mutant PAM-3 protein detected by the COOH-terminal domain antibody were almost equal (densitometric values differed by <10% of wild type PAM-3, n = 2), indicating that the PHM specific activities of all three PAM-3 proteins were very similar. The ratios of PAL to PHM activity for PAM-3-KDEL and PAM-3-KDEV (1.5) were indistinguishable from each other (p < 0.05, n = 4) but were significantly less than the PAL:PHM ratio of wild type PAM-3 (PAL:PHM 2; p < 0.05, n = 4). Addition of the COOH-terminal KDEL/KDEV tetrapeptide does not cause more than a slight decrease of PAL activity. Thus we conclude that the ER/cis-Golgi network provides an environment sufficient for the maturation of fully active PAM-3 protein.


Figure 5: PAM-3-KDEL protein expressed in hEK-293 cells. Extracts of hEK-293 cells expressing wild type PAM-3 (29), PAM-3-KDEL, or PAM-3-KDEV were assayed for PHM and PAL activities. A, samples of each extract containing equal amounts of PHM activity (1 nmol/h) were analyzed by immunoblot with monoclonal antibody to the COOH-terminal domain of PAM (antibody 6E6) and ECL. B, PAL activity/1 nmol/h of PHM activity for each cell extract; errorbars show standard errors obtained from quadruplicate assays. Similar data were observed on two separate sets of extracts. The specific activities of PHM and PAL in extracts of wild type hEK-293 cells were less than 1% of those activities in the transfected cells (29). WT, wild type.




DISCUSSION

When PAM proteins were expressed in neuroendocrine cells (i.e. AtT-20) or in non-neuroendocrine cells lacking a regulated secretory pathway (i.e. hEK-293), active PHM and PAL were produced(23, 29) . These findings suggest that the regulated secretory pathway and secretory granules are not necessary for the maturation of active PAM proteins. For example, the 10 amino acid pro-region of PAM, which is well conserved in evolution, is removed in a post-TGN compartment in AtT-20 cells and is not removed in hEK-293 cells; PHM activity is unaffected by the presence of the pro-region (34). We wanted to identify the compartment in which maturation of PAM occurred. In this study, we created a mutant soluble bifunctional PAM protein with the COOH-terminal KDEL ER retention/retrieval signal (PAM-3-KDEL) to examine the effect of ER retention on enzyme activities. Based on immunofluorescence staining, pulse-chase experiment and Endo H sensitivity, the PAM-3-KDEL protein was efficiently retained in the ER/cis-Golgi network. The ER retention of PAM-3-KDEL was mediated by the genuine KDEL ER retention/retrieval mechanism since attachment of an inactive homologue, KDEV, to PAM-3 had little effect on trafficking of PAM-3.

COOH-terminal attachment of the KDEL sequence was sufficient to cause extremely efficient retention/retrieval of PAM-3. In other secretory proteins examined after addition of the KDEL sequence, inefficient ER retention was often observed(35, 36) . Proteins that are efficiently retained in the ER are often distinguished by the presence of acidic amino acid residues upstream of the KDEL sequence(37, 38) . Addition of an acidic amino acid sequence found in an ER resident protein (i.e. protein disulfide isomerase) upstream of a COOH-terminal KDEL signal enhanced the ER retention efficiency of the secretory protein, interleukin-6(36) . Thus certain features of upstream sequence, such as acidic residues, may determine the efficiency of recognition by the KDEL receptor, and the acidic residues in the COOH-terminal 30 amino acid region of PAM-3 (11 of 30 residues are Asp or Glu) (Fig. 1) may contribute to the efficient ER retention observed for the PAM-3-KDEL protein.

During passage through the secretory pathway, newly synthesized PAM proteins undergo extensive post-translational processing. Signal sequence removal and N-linked glycosylation occur in the ER (25). As PAM proteins travel through the Golgi stacks, O-linked glycosylation, sialylation, and Tyr- or O-linked oligosaccharide sulfation take place(26, 27) . In transfected AtT-20 cells, all of the post-signal peptide cleavages of PAM occur in a post-TGN compartment(18, 23) . These endoproteolytic cleavages have a modulatory effect on the PHM activity of PAM-3 and PAM-1 (34, 39) but are not essential for the generation of activity. Other peptide-processing enzymes such as furin(13, 21) , PC1, and PC2 (17, 19), which are synthesized as proenzymes, are inactive until activated by post-translational events following intra- (i.e. furin) or intermolecular autocatalytic cleavage in the ER. The 10-amino acid NH-terminal pro-region of PAM is not required during the biosynthesis of PAM, and its removal has little or no effect on PHM activity(34) . A truncated, soluble form of furin retained in the ER by addition of a COOH-terminal KDEL sequence underwent propeptide cleavage but was not active on exogenous substrates coexpressed in vivo(16, 21) . Thus in the case of furin, pro-region cleavage in the ER seems to be a prerequisite to proceed to further post-ER events for complete maturation(21) . Results of this study showing enzymatically active PAM-3-KDEL protein in the ER ( Fig. 4and Fig. 5) suggest that post-translational events occurring in the Golgi and later compartments are not required for maturation of active PAM-3.

Newly synthesized PAM proteins are catalytically competent in the ER. It seems unlikely that the peptidylglycine substrate acted upon by PAM would be available in the ER. Endo- and exoproteolytic cleavages of peptide precursors are required to reveal COOH-terminal Gly residues in most prohormones, and these cleavages occur first in the TGN and largely in secretory granules(1, 2) . Accordingly, compartments in which peptide amidation occur are restricted to the TGN and secretory granules by the availability of substrates as seen for the proopiomelanocortin-derived joining peptide amide immunoreactivity in the TGN and secretory granules(10) . It is not clear whether PAM-3-KDEL could -amidate a peptidylglycine substrate presented to it in the ER. Our assays were always carried out after the addition of exogenous copper and ascorbate and do not address the issue of when copper is loaded onto PHM or when ascorbate becomes available to the enzyme. In addition, the lumenal pH in the ER is not as close to the optimal pH (5.5-6) of PAM as the lumenal pH in the TGN and immature secretory granules. Thus, this study strongly supports the hypothesis that restricted localization of peptide amidation is determined by the restricted activation of endo- and exoproteases along with the lumenal milieu rather than by post-translational activation of PAM.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant DK-32949. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Neuroscience, Wood Basic Science Bldg., Room 907, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-6921; Fax: 410-955-0681; E-mail: eipper lab@qmail.bs.jhu.edu.

The abbreviations used are: TGN, trans-Golgi network; PAM, peptidylglycine -amidating monooxygenase; PHM, peptidylglycine -hydroxylating monooxygenase; PAL, peptidyl--hydroxyglycine -amidating lyase; DMEM/F-12, Dulbecco's modified Eagle's medium and nutrient mixture F-12 mixed in equal volumes; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminiscence; ER, endoplasmic reticulum; Endo H, endoglycosidase H.

S. L. Milgram, R. E. Mains, and B. A. Eipper, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Dick Mains, Luc Paquet, and Sharon Milgram for critically reading this manuscript. The monoclonal antibody to PAM COOH-terminal domain was prepared in collaboration with Dr. Sharon Milgram. We also thank Zina Garrett for secretarial assistance, Carla Berard for help with tissue culture, and Marie Bell for general laboratory assistance.


REFERENCES
  1. Sossin, W. S., Fisher, J. M., and Scheller, R. H.(1989) Neuron2, 1407-1417 [Medline] [Order article via Infotrieve]
  2. Steiner, D. F.(1991) in Peptide Biosynthesis and Processing (Fricker, L. D., ed) pp. 1-15, CRC Press, Inc., Boca Raton, FL
  3. Eipper, B. A., Stoffers, D. A., and Mains, R. E.(1992) Annu. Rev. Neurosci.15, 57-85 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bradbury, A. F., and Smyth, D. G.(1991) Trends Biochem. Sci.16, 112-115 [CrossRef][Medline] [Order article via Infotrieve]
  5. Eipper, B. A., Perkins, S. N., Husten, E. J., Johnson, R. C., Keutmann, H. T., and Mains, R. E.(1991) J. Biol. Chem.266, 7827-7833 [Abstract/Free Full Text]
  6. Perkins, S. N., Husten, E. J., and Eipper, B. A.(1990) Biochem. Biophys. Res. Commun.171, 926-932 [Medline] [Order article via Infotrieve]
  7. Takahashi, K., Okamoto, H., Seino, H., and Noguchi, M.(1990) Biochem. Biophys. Res. Commun.169, 524-530 [Medline] [Order article via Infotrieve]
  8. Young, S. D., and Tamburini, P. P.(1989) J. Am. Chem. Soc.111, 1933-1934
  9. Katopodis, A. G., Ping, D., and May, S.(1990) Biochemistry29, 6115-6120 [Medline] [Order article via Infotrieve]
  10. Schnabel, E., Mains, R. E., and Farquhar, M. G.(1989) Mol. Endocrinol.3, 1223-1235 [Abstract]
  11. Fisher, J. M., and Scheller, R. H.(1988) J. Biol. Chem.263, 16515-16518 [Free Full Text]
  12. Anderson, R. G. W., and Orci, L.(1988) Cell Biol.106, 539-543
  13. Leduc, R., Molloy, S. S., Thorne, B. A., and Thomas, G.(1992) J. Biol. Chem.267, 14304-14308 [Abstract/Free Full Text]
  14. Goodman, L. J., and Gorman, C. M.(1994) Biochem. Biophys. Res. Commun.201, 795-804 [CrossRef][Medline] [Order article via Infotrieve]
  15. Matthews, G., Shennan, K. I. J., Seal, A. J., Taylor, N. A., Colman, A., and Docherty, K.(1994) J. Biol. Chem.269, 588-592 [Abstract/Free Full Text]
  16. Rehemtulla, A., Dorner, A. J., and Kaufman, R. J.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 8235-8239 [Abstract]
  17. Vindrola, O., and Lindberg, I.(1993) Mol. Endocrinol.6, 1441-1450 [Abstract]
  18. Milgram, S. L., and Mains, R. E.(1994) J. Cell Sci.107, 737-745 [Abstract/Free Full Text]
  19. Benjannet, S., Rondeau, N., Paquet, L., Boudreault, A., Lazure, C., Chrétien, M., and Seidah, N. G.(1993) Biochem. J.294, 735-743 [Medline] [Order article via Infotrieve]
  20. Zhou, A., and Mains, R. E.(1994) J. Biol. Chem.269, 17440-17447 [Abstract/Free Full Text]
  21. Molloy, S. S., Thomas, L., VanSlyke, J. K., Stenberg, P. E., and Thomas, G.(1994) EMBO J.13, 18-33 [Abstract]
  22. Vey, M., Schäfer, W., Berghöfer, S., Klenk, H.-D., and Garten, W.(1994) J. Cell Biol.127, 1829-1842 [Abstract]
  23. Milgram, S. L., Johnson, R. C., and Mains, R. E.(1992) J. Cell Biol.117, 717-728 [Abstract]
  24. Oyarce, A. M., and Eipper, B. A.(1993) J. Neurochem.60, 1105-1114 [Medline] [Order article via Infotrieve]
  25. Yun, H.-Y., Johnson, R. C., Mains, R. E., and Eipper, B. A.(1993) Arch. Biochem. Biophys.301, 77-84 [CrossRef][Medline] [Order article via Infotrieve]
  26. Yun, H.-Y., Keutmann, H. T., and Eipper, B. A.(1994) J. Biol. Chem.269, 10946-10955 [Abstract/Free Full Text]
  27. Maltese, J.-Y., and Eipper, B. A.(1993) Endocrinology133, 2579-2587 [Abstract]
  28. Andres, D. A., Rhodes, J. D., Meisel, R. L., and Dixon, J. E.(1991) J. Biol. Chem.266, 14277-14282 [Abstract/Free Full Text]
  29. Tausk, F. A., Milgram, S. L., Mains, R. E., and Eipper, B. A.(1992) Mol. Endocrinol.6, 2185-2196 [Abstract]
  30. Husten, E. J., and Eipper, B. A.(1991) J. Biol. Chem.266, 17004-17010 [Abstract/Free Full Text]
  31. Milgram, S. L., Mains, R. E., and Eipper, B. A.(1993) J. Cell Biol.121, 23-26 [Abstract]
  32. Milgram, S. L., Eipper, B. A., and Mains, R. E.(1994) J. Cell Biol.124, 33-41 [Abstract]
  33. Lodish, H. F., Kong, N., Snider, M., and Strous, G. J. A. M.(1983) Nature304, 80-83 [Medline] [Order article via Infotrieve]
  34. Husten, E. J., Tausk, F. A., Keutmann, H. T., and Eipper, B. A.(1993) J. Biol. Chem.268, 9709-9717 [Abstract/Free Full Text]
  35. Rose-John, S., Schooltink, H., Schmitz-Van de Leur, H., Müllberg, J., Heinrich, P., and Graeve, L.(1993) J. Biol. Chem.268, 22084-22091 [Abstract/Free Full Text]
  36. Zagouras, P., and Rose, J. K.(1989) J. Cell Biol.109, 2633-2640 [Abstract]
  37. Munro, S., and Pelham, H. R. B.(1987) Cell48, 899-907 [Medline] [Order article via Infotrieve]
  38. Denecke, J., De Ryke, R., and Botterman, J.(1992) EMBO J.11, 2345-2355 [Abstract]
  39. Husten, E. J., and Eipper, B. A.(1994) Arch. Biochem. Biophys.312, 487-492 [CrossRef][Medline] [Order article via Infotrieve]

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