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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4521-4531
Copyright © 2004 by The Endocrine Society

Differential Cellular Handling of Defective Arginine Vasopressin (AVP) Prohormones in Cells Expressing Mutations of the AVP Gene Associated with Autosomal Dominant and Recessive Familial Neurohypophyseal Diabetes Insipidus

Jane H. Christensen, Charlotte Siggaard, Thomas J. Corydon, Gary L. Robertson, Niels Gregersen, Lars Bolund and Søren Rittig

Pediatric Research Laboratory (J.H.C.), Department of Pediatrics (C.S., S.R.), and Research Unit for Molecular Medicine (N.G.), Aarhus University Hospital, DK-8200 Aarhus N, Denmark; Department of Human Genetics (T.J.C., L.B.), University of Aarhus, DK-8000 Aarhus C, Denmark; and Department of Medicine (G.L.R.), Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Søren Rittig, M.D., Department of Pediatrics, Aarhus University Hospital, Skejby Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. E-mail: rittig{at}iekf.au.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An unusual mutation in the arginine vasopressin (AVP) gene, predicting a P26L amino acid substitution of the AVP prohormone, is associated with autosomal recessive familial neurohypophyseal diabetes insipidus (FNDI). To investigate whether the cellular handling of the P26L prohormone differed from that of the Y21H prohormone associated with autosomal dominant inheritance of FNDI, the mutations were examined by heterologous expression in cell lines. Immunoprecipitation demonstrated retarded processing and secretion of the Y21H prohormone, whereas the secretion of the P26L prohormone seemed to be unaffected. Confocal laser scanning microscopy showed accumulation of the Y21H prohormone in the endoplasmic reticulum, whereas the P26L prohormone and/or processed products were localized in secretory granules in the cellular processes. RIA analysis showed reduced amounts of immunoreactive Y21H-AVP and P26L-AVP in the cell culture medium. Thus, the recessive mutation does not seem to affect the intracellular trafficking but rather the final processing of the prohormone. Our results provide an important negative control in support of the hypothesis that autosomal dominant inheritance of FNDI is caused by mutations in the AVP gene that alter amino acid residues important for folding and/or dimerization of the neurophysin II moiety of the AVP prohormone and subsequent transport from the endoplasmic reticulum.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOSOMAL DOMINANT FAMILIAL neurohypophy-seal diabetes insipidus (adFNDI) is characterized by polyuria caused by a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP) (1). The AVP deficiency seems to develop gradually during the first few years of life (2, 3, 4) and is probably due to degeneration of the AVP-producing magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus (3, 5). Until now, the disease has been linked to 50 different mutations in the AVP gene encoding the AVP preprohormone (4, 6, 7). The AVP preprohormone comprises a signal peptide (codons 1–19), the AVP nonapeptide moiety (codons 20–28), the neurophysin II domain (codons 32–124), and a C-terminal glycopeptide, copeptin (codons 126–164) [numbered according to the AVP gene sequence (8) and GenBank accession no. M11166)]. Most of the mutations associated with adFNDI are located, not in the part of the gene encoding the AVP nonapeptide, but rather in the parts encoding the neurophysin II domain of the AVP prohormone. Virtually all of them affect amino acid residues known or predicted to be important for folding and/or dimerization of the neurophysin II domain.

This has led to the hypothesis (9, 10) that the mutations lead to the production of a mutant hormone precursor that fails to fold and/or dimerize efficiently in the endoplasmic reticulum (ER) and, as a consequence, is retained by the ER protein quality control machinery resulting in cytotoxic accumulation and/or aggregation of protein in the neurons. Heterologous expression experiments in cell cultures and transgenic animals support, at least partly, the hypothesis by concurrently showing that adFNDI mutations lead to the production of a mutant AVP prohormone that, compared with the wild type (WT), is processed inefficiently into AVP and neurophysin II, probably as a result of its retention in the ER (11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Toxic effects on the differentiation or long-term viability of neuronal cell types have been observed in some cases (9, 12), and recently it has been demonstrated that the progressive development of a typical diabetes insipidus (DI) phenotype in a murine knock-in model of an adFNDI mutation was accompanied with an induction of the ER resident chaperone, Grp78, and a progressive loss of the AVP-producing neurons in the paraventricular and supraoptic nuclei (21). In the same study, however, a murine knock-in model of another adFNDI mutation, which is associated with a mild phenotype and delayed onset in humans, does not develop any apparent DI phenotype.

An important deficiency in prior studies is a negative control confirming that AVP gene mutations that alter amino acid residues that are not essential for folding of the AVP prohormone also do not result in adFNDI by leading to the production of mutant precursors that are retained in the ER. Recently a 301C>T mutation in the AVP gene (numbered according to Ref. 8 and GenBank accession no. M11166) predicting a substitution of the seventh amino acid residue of the hormone moiety of the AVP prohormone (proline 26 to leucine, P26L) has been identified in a kindred with autosomal recessive FNDI (arFNDI) (22). Despite the fact that this mutation is situated in close proximity to an adFNDI mutation predicting a substitution of the second amino acid residue of the hormone moiety (tyrosine 21 to histidine, Y21H) (23), it causes clinically significant deficiency in antidiuretic function only in the homozygous state. In addition, it looks as if the mutation does not result in degeneration of the neurohypophysis in view of the fact that relative large amounts of immunoreactive P26L-AVP appear to be produced in the homozygous state, and one of the three affected children was reported to have a normal posterior pituitary bright spot on magnetic resonance images (22). These clinical differences as well as the fact that P26 has never been identified as important for folding of the AVP prohormone suggest that the P26L prohormone, unlike the Y21H prohormone associated with adFNDI (20), is folded, dimerized, and transported unhindered from the ER.

The object of the present study was to determine whether these two mutations have different effects on the cellular processing of the AVP prohormone. Differences should be obvious because the mutations are associated with, respectively, a recessive phenotype with no apparent manifestation in heterozygous individuals and a classical adFNDI phenotype. To test this prediction, we compared the cellular handling of P26L and Y21H prohormones by heterologous expression in neurogenic and neuronal cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of expression constructs

PcDNA1-WT and pcDNA3.1-WT denote, respectively, a pcDNA1 or pcDNA3.1(+) vector (Invitrogen, Groningen, The Netherlands) containing the 495-bp coding region of a human AVP cDNA (a kind gift from Professor D. Richter, Eppendorf University, Hamburg, Germany) corrected for a discrepancy from the normal genomic sequence (13). Using pcDNA1-WT as a template, a mutation corresponding to the 285T>C substitution of the AVP gene previously described (23) was introduced by PCR-based site-directed mutagenesis (24). Another mutation corresponding to the 301C>T substitution previously described (22) was introduced in a similar fashion. The resulting DNA fragments (218 and 212 bp, respectively) were used as antisense megaprimers in combination with a vector-specific sense primer in a second round of PCR amplification. The resulting DNA fragments (352 bp) were cleaved with the restriction endonucleases, HindIII and XmaI (New England Biolabs, Inc., Beverly, MA) and cloned into pcDNA1-WT. Finally, the complete mutated cDNAs were excised from the pcDNA1 vector by cleavage with HindIII and XbaI (New England Biolabs) and cloned into the multiple cloning site of an empty pcDNA3.1(+) vector (Invitrogen). The resulting vectors were named pcDNA3.1-Y21H and pcDNA3.1-P26L. To verify the presence of the mutations introduced and exclude other mutations, the nucleotide sequence of the AVP cDNA coding region as well as its flanking regions was determined by sequencing in both directions using a dRhodamine terminator cycle sequencing kit with AmpliTaq DNA polymerase, fluorescent sequencing, and an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA). All primer sequences and PCR conditions are available on request.

Cell culture and transfection

Human teratocarcinoma NTera2/D1 cells (Stratagene, La Jolla, CA) and mouse neuroblastoma Neuro2A cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM, high glucose formulation (GIBCO BRL, Life Technologies Ltd., Paisly, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO, Life Technologies), 10,000 IU/ml penicillin, and 1% streptomycin (both Leo Pharmaceutical Products Ltd. A/S, Ballerup, Denmark). The cells were maintained in a 5% CO2 atmosphere at 37 C. Transfection with pcDNA3.1-WT, -Y21H, -P26L, or empty pcDNA3.1(+) vector as a control was performed on 50% confluent cells using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Hvidovre, Denmark) as previously described (25). Unless indicated, human teratocarcinoma NTera2/D1 cells were used.

Metabolic labeling and immunoprecipitation

Cells were plated in six-well tissue culture test plates (9 cm2) (Techno Plastic Products, Trasadingen, Switzerland) for metabolic labeling and immunoprecipitation. Twenty-four hours after transfection, cells were incubated in 1 ml RPMI 1640 (without L-cysteine and L-methionine) (BioWhittaker, Walkersville, MD), supplemented with 4 mg/ml L- cysteine, 1 mg/ml L-methionine, 10% heat-inactivated FCS, 10,000 IU/ml penicillin, 1% streptomycin, and 0.1 mCi Pro-mix L-[35S] in vitro labeling mix (Amersham Pharmacia Biotech, Little Chalfont, UK) for 18 h. For pulse-chase experiments, the intracellular pool of cysteine and methionine was depleted from the cells before labeling by incubation in 500 µl medium (without L-cysteine, L-methionine, and in vitro labeling mix) for 30 min. Cells were pulsed for 30 min in 500 µl RPMI 1640 (without L-cysteine and L-methionine), supplemented with 10% heat-inactivated FCS, 10,000 IU/ml penicillin, 1% streptomycin, and 0.1 mCi Pro-mix L-[35S] in vitro labeling mix and chased for the times indicated in DMEM, high glucose formulation, supplemented with 10% heat-inactivated FCS, 10,000 IU/ml penicillin, 1% streptomycin, 0.1% L-cysteine, and 0.1% L-methionine. Cell culture medium was harvested and cells were lysed with 50 mM Tris (pH 8.0) containing 0.5% deoxycholic acid, 150 mM NaCl, 1% triton X-100, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 Kallikrein Inhibitor Units (KIU)/ml Trasylol (Bayer AG, Leverkusen, Germany), 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 10 U/ml bacitracin.

To remove unrelated proteins, samples were incubated with Protein A Sepharose CL-4B (Amersham Pharmacia Biotech) and rabbit IgG (Sigma BioSciences, St. Louis, MO) at 4 C for 1 h. After a brief centrifugation (210 x g), the AVP gene products were immunoprecipitated from the sample supernatants by incubation at 4 C overnight with Protein A Sepharose CL-4B and either rabbit polyclonal anti-neurophysin II or anti-vasopressin antiserum (ICN Biomedicals, Inc., Costa Mesa, CA). After repeated rounds of washing, immunocomplexes were released from the Protein A Sepharose CL-4B beads by incubation in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) (containing 350 mM dithiothreitol) at 95 C for 5 min and separated on 15% Tris-HCl gels (Bio-Rad Laboratories) by SDS-PAGE. Proteins were visualized by Bio-Safe Coomassie blue staining (Bio-Rad Laboratories) to control whether each sample contained equal amounts of antiserum. Visualization and densitometric quantification of radioactivity was performed using STORM 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software, version 4.2 (Molecular Dynamics). When indicated and before separation, immunoprecipitates were treated with 0 or 250 U endoglycosidase H (Endo H; New England Biolabs) for 2 h at 37 C according to the manufacturer’s instructions. Untreated samples of cell lysate (5 µl) were separated by SDS-PAGE (as described above), and proteins were visualized by Bio-Safe Coomassie blue staining to control whether equal amounts of total cell protein were extracted from the cells. Radioactivity was visualized using the PhosphorImager (Molecular Dynamics) and ImageQuant software (Molecular Dynamics) to control whether equal amounts of label were incorporated into the proteins.

Immunostaining and microscopy

For immunostaining and confocal laser scanning microscopy, NTera2/D1 or Neuro2A cells were plated in Nunc SlideFlasks (10 cm2) (Nalge Nunc International, Rochester, NY). To induce differentiation to neuronal cells, the culture medium was exchanged with serum-free medium 24 h after transfection. After an additional 48 h, the cells were fixed in 4% formaldehyde freshly prepared from paraformaldehyde, permeabilized in ice-cold 70% ethanol, and hydrated in PBS. A two-layer immunostaining procedure was performed essentially as described (13) using a primary rabbit anti-neurophysin II antiserum (1:300 for NTera2/D1 cells and 1:50 for Neuro2A cells) (ICN Biomedicals) and a secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (1:400) (Molecular Probes, Inc., Eugene, OR). Colocalization with the ER or secretory granules was detected by costaining with either mouse anti-KDEL (1:100) (StressGen Biotechnologies Corp., San Diego, CA), recognizing Grp78 and Grp94, or anti-Chromogranin A (1:100) (NeoMarkers, Fremont, CA) monoclonal primary antibodies and a secondary Alexa Fluor 568 goat anti-mouse IgG antibody (1:400) (Molecular Probes). Visualization was performed by confocal laser scanning microscopy (Leica Microsystems AG, Wetzlar, Germany).

RIA measurements

Cells were plated in either 6-well tissue culture testplates (9 cm2) (Techno Plastic Products) for RIA measurement of AVP immunoreactivity (irAVP) in the culture medium or tissue culture flasks (75 cm2) (Techno Plastic Products) for combined irAVP measurement and Northern blot analysis. Cells were supplied with fresh culture medium 24 h after transfection, and after an additional 24 h culturing, medium was collected and cells were harvested. Culture medium was either analyzed directly or extracted before analysis on Sep-Pak Plus C18 extraction cartridges (Waters Corp., Milford, MA) according to the manufacturer’s instructions. Extracts were dissolved in culture medium collected from cells transfected with empty pcDNA3.1(+) vector to make them equal unextracted samples. RIA was performed as described (26) using an AVP antibody (10169) produced by Dr. Jacques Dürr (27) at a final dilution of 1:1,000,000. Cross-reactivity of the antibody at 50% binding has previously been determined for a number of analogous peptides: [Lys8]-vasopressin 30%, [Arg8]-vasotocin less than 1.6%, [deamino-Cys1, D-Arg8]-vasopressin less than 1.2%, [Lys8]-vasotocin less than 0.8%, and oxytocin less than 0.005%. Assay cross-reactivity against Y21H-AVP and P26L-AVP was determined in two independent experiments (data not shown) using chemically synthesized peptides (KJ Ross-Petersen A/S, Hørsholm, Denmark) identical to authentic AVP except for the Y21H or P26L substitutions. Because there was no significant difference in the cross-reaction against Y21H-AVP, P26L-AVP, and normal AVP at peptide concentrations ranging from 0.0 to 1.2 pg/ml (as determined by comparison of the slopes of their individual logit transformed standard curves (28, 29)], irAVP in the medium was measured without adjustment. To adjust for differences in cell culture size, values were expressed per milligram of total cell lysate protein as determined by the DC protein assay (Bio-Rad Laboratories) (30).

Northern blot analysis

Total RNA was isolated from transfected cells using the TRIZOL Reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA (7.5 µg/lane) was separated by electrophoresis in a 1% agarose formaldehyde gel and blotted to a Zeta Probe nylon membrane (Bio-Rad Laboratories) by capillary blotting. A gel-purified 580-bp HindIII and XbaI fragment of pcDNA3.1-WT was used as an AVP mRNA-specific probe. An RT-PCR amplified glyceraldehyde-3-phosphate dehydrogenase mRNA-specific probe (cDNA position 53–417, 365 bp) was used as an internal control. Probes were labeled with [{alpha}-32P]dATP (3000 Ci/mmol) by random priming using Prime-It II random primer labeling kit (Stratagene) and subsequently purified on ProbeQuant G50 microcolumns (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer’s instructions. Probe hybridization was performed according to standard laboratory procedures. Visualization and densitometric quantification of radioactivity were performed using the PhosphorImager (Molecular Dynamics) and ImageQuant software (Molecular Dynamics).

Statistical analysis

The slopes of the logit transformed Y21H-AVP, P26L-AVP, and normal AVP standard curves were compared by linear regression analysis. Amounts of irAVP in the cell culture medium were compared by Student’s t test. P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AVP biosynthesis

To investigate the intracellular AVP biosynthesis, transiently transfected NTera2/D1 cells were metabolically labeled and the proteins immunoprecipitated with antineurophysin II or anti-vasopressin antiserum before size separation by reducing SDS-PAGE and visualization (Figs. 1–3GoGoGo).



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  		FIG. 1. SDS-PAGE analyses of immunoprecipitates derived from continuously labeled NTera2/D1 cells. Cells transiently transfected with pcDNA3.1-WT, -Y21H, -P26L, or empty pcDNA3.1 vector were metabolically labeled for 18 h. Cell lysates and medium were collected, and immunoreactive proteins were precipitated before separation on 15% Tris-HCl gels by SDS-PAGE and visualization using the PhosphorImager and ImageQuant software. Immunoprecipitation was performed with rabbit polyclonal anti-neurophysin II antiserum (A) or rabbit polyclonal anti-neurophysin II (NPII) or anti-vasopressin (VP) antiserum (B), as indicated above the lanes. The migration of molecular weight standards is indicated on the left side of the figures. The presumed identities of the precipitated products are indicated on the right side of the figures. The results are representative of three independent experiments.

 


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  		FIG. 2. SDS-PAGE analyses of Endo H-treated immunoprecipitates derived from continuously labeled NTera2/D1 cells. Cells transiently transfected with pcDNA3.1-WT, -Y21H, -P26L, or empty pcDNA3.1 vector were metabolically labeled for 18 h. Cell lysates (A) and medium (B) were collected, and immunoreactive proteins were precipitated with rabbit polyclonal anti-neurophysin II antiserum and treated with 0 (–) or 250 U (+) of Endo H before separation on 15% Tris-HCl gels by SDS-PAGE and visualization using the PhosphorImager and ImageQuant software. Asterisks (B) indicate the migration of two unrelated proteins appearing in the samples when pretreatment of the samples with Protein A Sepharose CL-4B and rabbit IgG was omitted. The migration of molecular weight standards is indicated on the left side of the figure. The presumed identities of the precipitated products are indicated on the right side of the figures. The results are representative of two independent experiments.

 


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  		FIG. 3. SDS-PAGE analyses of immunoprecipitates derived from pulse chase-labeled NTera2/D1 cells. Cells transiently transfected with pcDNA3.1-WT (A), pcDNA3.1Y21H (B), or pcDNA3.1-P26L (C) were pulse labeled metabolically for 30 min and chased for the times indicated above the lanes. Cell lysates and medium were collected and immunoprecipitated with rabbit polyclonal anti-neurophysin II antiserum before separation on 15% Tris-HCl gels by SDS-PAGE and visualization using the PhosphorImager and ImageQuant software. Cell lysates and medium from cells transfected with empty pcDNA3.1 vector were analyzed in parallel as controls (data not shown). The migration of molecular weight standards is indicated on the left side of the figures. The presumed identities of the precipitated products are indicated on the right side of the figures.

 
Immunoprecipitation of lysates from continuously labeled (18 h) cells transfected with pcDNA3.1-WT with anti-neurophysin II yielded four different protein bands (Fig. 1Go, A and B, lane 1). The relative mobility of these bands was similar to those predicted for AVP prohormone that was glycosylated (>15.4 kDa), unglycosylated (15.4 kDa), partially processed to vasopressin-neurophysin II (11.2 kDa), and neurophysin II (9.8 kDa), respectively. Immunoprecipitation of cell culture medium with anti-neurophysin II yielded two bands with mobilities similar to those predicted for glycosylated prohormone (>15.4 kDa) and completely processed neurophysin II (9.8 kDa) (Fig. 1AGo, lane 5, and 1B, lane 3). The reducing conditions of the analyses did not allow the detection of possible dimers.

The bands presumed to be glycosylated prohormone were also immunoprecipitated from the lysates and the medium with anti-vasopressin (Fig. 1BGo, lanes 2 and 4), confirming that they contained the hormone moiety. The band presumed to be partially processed vasopressin-neurophysin II, and thus assumed to contain the hormone moiety, was not immunoprecipitated in any detectable amounts with anti-vasopressin (Fig. 1BGo, lane 2). However, this was probably a result of the relatively lower potency of this antiserum, compared with that of the anti-neurophysin II antiserum.

When cell lysates were immunoprecipitated with anti-neurophysin II and subsequently treated with Endo H, which cleaves the N-linked oligosaccharides attached in the ER but not those modified in the medial-Golgi network, the band presumed to be glycosylated prohormone (Fig. 2AGo, lane 1) shifted almost completely toward the mobility of the presumed unglycosylated form (Fig. 2AGo, lane 2) indicating that the majority of the band represents glycosylated prohormone that has been N-glycosylated in the ER but not yet modified in the medial-Golgi network. However, the band immunoprecipitated from the medium and presumed to represent glycosylated prohormone (Fig. 2BGo, lane 1) was almost Endo H resistant (Fig. 2BGo, lane 2), indicating that it represent prohormone that has been terminally glycosylated as a result of its transport through the Golgi complex. Furthermore, the band presumed to be terminally glycosylated prohormone (Fig. 1AGo, lane 5) had a slightly lower relative mobility than that presumed to be core glycosylated prohormone (Fig. 1AGo, lane 1), which should reflect the increase in molecular weight obtained in the Golgi complex on oligosaccharide modification.

In general, the migration positions of the different protein bands diverged somewhat from previous published experiments (e.g. Ref. 17). This could reflect differences between cell lines (human teratocarcinoma NTera2/D1 cells vs. rat adrenal pheochromocytoma PC12 cells transfected with mouse prohormone convertase 2). However, it could also be the result of other experimental variations because different migration positions have been published even within the same cell line [mouse neuroblastoma Neuro2A cells (12, 14)].

Lysates and medium from cells transfected with pcDNA3.1-P26L yielded immunoprecipitable products that were similar to those derived from cells transfected with pcDNA3.1-WT (Fig. 1AGo, lanes 3 and 7). Densitometric quantification of the radioactivity immunoprecipitated from the cell culture medium in three independent experiments revealed that the relative amounts (compared with WT) of presumed glycosylated prohormone and processed neurophysin II were, respectively, 0.97 ± 0.05 (mean ± SD) and 0.52 ± 0.04. These results indicate that the secretion of processed neurophysin II is reduced, compared with WT, whereas the secretion of the glycosylated prohormone seems to be unaffected.

Immunoprecipitation of lysates from cells transfected with the pcDNA3.1-Y21H with anti-neurophysin II also yielded two bands that migrated with mobilities similar to those expected for glycosylated and unglycosylated prohormone but no detectable amounts of any other material (Fig. 1AGo, lane 2). Immunoprecipitation of the medium yielded two bands with relative mobilities corresponding to those expected for glycosylated prohormone and fully processed neurophysin II (Fig. 1AGo, lane 6). The relative amounts (compared with WT) of presumed glycosylated prohormone and processed neurophysin II were, respectively, 0.10 ± 0.02 and 0.37 ± 0.06, indicating that the secretion of both forms is reduced, compared with WT

The effects of Endo H treatment of anti-neurophysin II immunoprecipitated cell lysates and medium from cells transfected with either pcDNA3.1-Y21H or -P26L were similar to those observed for pcDNA3.1-WT (Fig. 2AGo, lanes 3–6, and 2B, lanes 3–6), indicating that neither WT nor mutant glycosylated prohormone is retained in the cells on modification in the Golgi complex but rather seems to be either secreted immediately or processed. The relative amounts of presumed glycosylated products in these experiments (Fig. 2Go, A and B) (determined as described above) were consistent with the findings presented above (Fig. 1AGo); however, it was not possible to detect any processed products, indicating that the Endo H incubation procedure interferes with their stability.

To investigate the kinetics of the AVP biosynthesis, cells transfected with pcDNA3.1-WT, -Y21H, or -P26L were metabolically pulse labeled with [35S]-methionine and cysteine for 30 min; chased with unlabeled amino acids for 30 min or 1, 3, 6, or 24 h; and analyzed. When analyzed immediately after labeling (time zero), immunoprecipitation of lysates from cells transfected with pcDNA3.1-WT yielded a dense band with a mobility similar to that expected for glycosylated AVP prohormone and two lighter bands with higher mobility that should represent unglycosylated prohormone and processed vasopressin-neurophysin II (Fig. 3AGo, lane 1). The amounts of these intracellular products decreased progressively during the whole chase period (Fig. 3AGo, lanes 2–6). Almost undetectable amounts of a product that should represent neurophysin II were immunoprecipitated from the cell lysates after 30 min and 1 and 3 h of chase (Fig. 3AGo, lanes 2–4). Immunoprecipitation of the medium yielded trace amounts of the presumed terminally glycosylated prohormone after 30 min of chase (Fig. 3AGo, lane 7) and progressively larger amounts after 1 and 3 h (Fig. 3AGo, lanes 8 and 9), indicating that it was continuously released from the cells during this period. No product corresponding to vasopressin-neurophysin II was detected in the medium whereas trace amounts of a product that probably represented neurophysin II was immunoprecipitated from the medium after 1 h of chase (Fig. 3AGo, lane 8) and in larger amount after 3 h (Fig. 3AGo, lane 9), indicating that it was released from the cells almost immediately after its formation.

The kinetics of P26L prohormone synthesis and secretion was similar to that observed for the WT prohormone (Fig. 3Go, A and C). Immunoprecipitation of the cell lysates yielded appreciable amounts of a newly synthesized product, probably representing glycosylated, unprocessed prohormone, and considerable amounts of two other products, probably representing unglycosylated prohormone and partially processed vasopressin-neurophysin II (Fig. 3CGo, lane 1). The amounts of these products in the cell lysates decreased progressively with longer periods of chase (Fig. 3AGo, lanes 2–6). In addition, like the medium from the cells transfected with pcDNA3.1-WT, immunoprecipitation of the medium from the cells transfected with pcDNA3.1-P26L yielded progressively increasing amounts of two products, probably corresponding to glycosylated, unprocessed prohormone and completely processed neurophysin II (Fig. 3AGo, lanes 7–9). Densitometric quantification of the radioactivity immunoprecipitated from the cell culture medium in a single experiment revealed that the relative amounts of presumed processed neurophysin II (compared with the amounts of presumed unprocessed prohormone) were in average 0.5 times lower (range 0.4–0.7 for time points 30 min and 1, 6, and 24 h) than for cells transfected with pcDNA3.1-WT. This seems to be consistent with the results obtained with continuously labeled cells as presented in Fig. 1AGo in which the corresponding value was 0.58 ± 0.03 (mean ± SD), indicating that the proportion of presumed processed neurophysin II secreted from cells transfected with pcDNA3.1-P26L was smaller than from those transfected with pcDNA3.1-WT.

On the contrary, the kinetic pattern observed for the cells transfected with pcDNA3.1-Y21H differed appreciably from that of the cells transfected with pcDNA3.1-WT (or pcDNA3.1-P26L) (Fig. 3BGo). The amount of presumed processed vasopressin-neurophysin II, compared with the amount of presumed glycosylated prohormone, immunoprecipitated from the cell lysates immediately after labeling (time zero) was smaller than that derived from the cells transfected with pcDNA3.1-WT (compare Fig. 3Go, A and B, lane 1). Moreover, unlike the medium from these cells, immunoprecipitation of the medium from cells transfected with pcDNA3.1-Y21H did yield only trace amounts of the products presumed to be glycosylated prohormone or completely processed neurophysin II (Fig. 3BGo, lanes 10 and 11), indicating that neither of these products were released in appreciable amounts. Nevertheless, the amount of intracellular product attributable to glycosylated but unprocessed mutant prohormone decreased progressively after the initial period of labeling (Fig. 3BGo, lanes 1–6), indicating that it was either degraded or somehow rendered inaccessible. Densitometric quantification of the radioactivity immunoprecipitated from the cell culture medium in a single experiment at time points 6 and 24 h revealed that the relative amounts of presumed processed neurophysin II (compared with the amounts of presumed unprocessed prohormone) were on average 3.9 times higher (range 2.5–5.5) than for cells transfected with pcDNA3.1-WT. This seems also to be consistent with the results obtained with continuously labeled cells as presented in Fig. 1AGo in which the corresponding value was 4.00 ± 0.21 (mean ± SD), indicating that the proportion of presumed processed neurophysin II secreted from cells transfected with pcDNA3.1-Y21H was bigger than from those transfected with pcDNA3.1-WT.

In general, these findings indicate that, unlike the cells transfected with pcDNA3.1-Y21H, the cells transfected with pcDNA3.1-P26L synthesized, glycosylated, processed, and secreted the AVP prohormone with similar kinetics as the cells transfected with pcDNA3.1-WT.

Confocal laser-scanning microscopic analysis of the cellular localization of anti-neurophysin II reactive proteins

To compare the intracellular trafficking of WT and mutant prohormones and their eventual processed products, the intracellular localization of heterologously expressed neurophysin II-reactive proteins, and either endogenously expressed Grp78 and Grp94 or Chromogranin A was determined in transiently transfected Neuro2A cells (Fig. 4Go). In cells transfected with either pcDNA3.1-WT or pcDNA3.1-P26L, most of the neurophysin II reactive material appeared to be located in vesicular structures in the tips of the cellular processes (Fig. 4Go, A1 and A3) in which it colocalized with Chromogranin A (Fig. 4Go, D1 and D3), an established marker of secretory granules. However, some neurophysin II reactive protein was observed throughout the cell cytosol in which it colocalized partly in the perinuclear region with Grp78 and Grp94 (Fig. 4Go, C1 and C3), both ER resident proteins. In the cells transfected with pcDNA3.1-Y21H, the neurophysin II immunoreactivity was distributed largely in a peripheral band (Fig. 4Go, A2), colocalizing almost completely with the ER (Fig. 4Go, C2). A minor amount of neurophysin II reactive material appeared in the tips of the cellular processes but colocalization with Chromogranin A in this region was not apparent (Fig. 4Go, D2). In some cells, the neurophysin II reactive proteins were aggregated in large rounded perinuclear structures colocalizing with the ER (data not shown). The intracellular localization of heterologously expressed neurophysin II reactive proteins and endogenously expressed Grp78 and Grp94 in NTera2/D1 cells was similar to that observed within the Neuro2A cells (data not shown).



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  		FIG. 4. Cellular localization of neurophysin II reactive protein in transiently transfected Neuro2A cells as visualized by confocal laser-scanning microscopy. Cells were maintained until 72 h after transfection (the last 48 h in serum-free medium to induce differentiation to neuronal cells), fixed, and permeabilized. Heterologously expressed neurophysin II reactive proteins were detected by incubation with a rabbit anti-neurophysin II antiserum and a fluorescein-conjugated Alexa Fluor 488 goat anti-rabbit IgG antibody (green). Endogenously expressed Grp78 and Grp94, both ER resident proteins, were detected by a mouse anti-KDEL monoclonal antibody and a rhodamine-conjugated Alexa Fluor 568 goat anti-mouse IgG antibody (red). In other experiments endogenously expressed Chromogranin A, a marker of secretory granules, was detected by anti-Chromogranin A monoclonal antibody and a rhodamine-conjugated Alexa Fluor 568 goat antimouse IgG antibody (red). Colocalized proteins (yellow-orange) were detected by superimposing pictures of the same focal sections resulting in merging of the green and red. Row A, Pictures showing the cellular localization of neurophysin II reactive proteins; row B, pictures showing the cellular localization of endogenous expressed Grp78 and Grp94; row C, pictures from row A and B superimposed to show the degree of colocalization of neurophysin II reactive proteins and the ER (Grp78 and Grp94); row D, superimposed pictures showing the degree of colocalization of neurophysin II reactive proteins and secretory granules (Chromogranin A). Column 1, Cells transfected with pcDNA3.1-WT; column 2, pcDNA3.1-Y21H; and column 3, pcDNA3.1-P26L. Cells transfected with empty pcDNA3.1 vector were analyzed in parallel as a control (data not shown). The results shown are representative of cells examined in several separate experiments.

 
Secretion of irAVP into the cell culture medium

To determine whether the amount of irAVP secreted by NTera2/D1 cells transiently transfected with pcDNA3.1-WT, -Y21H, or -P26L differed significantly, culture medium was subjected to RIA analysis (Fig. 5Go, black columns). The amount (mean ± SD) of irAVP secreted by cells transfected with pcDNA3.1-WT was significantly greater (39.8 ± 2.1 pg per milligram cell protein per 24 h) than the amount secreted by cells transfected with either pcDNA3.1-Y21H (13.2 ± 0.8) or pcDNA3.1-P26L (10.3 ± 1.6). There were no statistically significant differences in the amounts of irAVP in the medium from cells transfected with independently produced DNA preparations (data not shown), and similar AVP mRNA levels were detected by Northern blot analysis (Fig. 5Go, inlet). Furthermore, other similar experiments using the same transfection procedures but either involving other cell types (data not shown) or expression vectors containing other cDNAs (20) have likewise demonstrated very little variability in mRNA levels. Thus, it is not likely that the differences observed (Fig. 5Go), which are highly reproducible in independent experiments, were due to any differences in the transcription or mRNA stability. To determine whether the irAVP produced by the cells comprised any unprocessed or partially processed prohormone, corresponding samples of cell culture medium were subjected to RIA analysis after extraction on Sep-Pak Plus C18 extraction cartridges (Fig. 5Go, shaded columns), a process eliminating all protein components of a molecular weight larger than 5–10 kDa. The amount of irAVP (mean ± SD) in extracted samples of culture medium from cells transfected with pcDNA3.1-WT (39.9 ± 1.7 pg per milligram cell protein per 24 h), pcDNA3.1-Y21H (12.6 ± 1.2), or pcDNA3.1-P26L (9.9 ± 1.6) was not significantly different from the amount measured in the corresponding untreated samples (Fig. 5Go, black columns). Thus, the extraction process did not eliminate any measurable amounts of irAVP, implying that no higher molecular weight forms of irAVP (i.e. unprocessed or partially processed prohormone) were detected in the cell culture medium by the RIA. However, this is probably because the RIA does not cross-react with such forms because the immunoprecipitation experiments indicate that the cells, at least those transfected with pcDNA3.1-WT or -P26L, release substantial amounts of unprocessed prohormone into the medium.



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  		FIG. 5. A, RIA measurements of irAVP secreted into the culture medium of NTera2/D1 cells. The amount of irAVP in the medium from cells transiently transfected with pcDNA3.1-WT, -Y21H, or -P26L was determined by RIA analysis of culture medium. Samples were either measured directly (black columns) or extracted on Sep-Pak Plus C18 extraction cartridges before analysis (shaded columns). Culture medium from cells transfected with empty pcDNA3.1 vector was analyzed in parallel as a control. All samples were analyzed in duplicate. The amount of irAVP secreted during 24 h relative to the total amount of cell protein in the incubated tissue culture test plates is given as mean ± SD from three wells expressed as picograms irAVP per milligram cell protein per 24 h. The results are representative of three independent experiments. B, Northern blot analysis of total RNA (7.5 µg/lane) from NTera2/D1 cells transiently transfected with pcDNA3.1-WT (lane 1), pcDNA3.1-Y21H (lane 2), pcDNA3.1-P26L (lane 3), and pcDNA3.1 vector (lane 4). The blot was incubated successively with [32P]-labeled AVP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA-specific probes as indicated. The results are representative of at least two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Y21H and P26L amino acid substitutions result from mutations in the AVP gene associated with, respectively, adFNDI and arFNDI. The clinical characteristics reported in affected members of the two kindreds in question are similar to some extent, including delayed onset of disease symptoms (1–3 and 2 yr of age, respectively) and complete correction of disease by treatment with the AVP analog, 1-desamino-8-D-AVP (22, 23). Despite these similarities, it is obvious that the pathogenesis of adFNDI and arFNDI is different because heterozygous carriers of the P26L mutation seem not to develop DI, and homozygous affected individuals apparently continue to secrete large amounts of immunoreactive P26L-AVP. In addition, the bright spot normally observed on T1-weighted magnetic resonance images of the posterior pituitary was reported to be present in one of the three children with arFNDI but was absent in all patients with overt adFNDI due the Y21H mutation and at least 14 other mutations of the AVP gene (Robertson, G. L., unpublished observations).

Because P26 has never been identified as important for folding of the AVP prohormone, it suggests that the P26L prohormone, unlike the Y21H prohormone, is folded, dimerized, and transported unhindered from the ER. To test this prediction, we compared the cellular handling of P26L and Y21H prohormones by heterologous expression in neurogenic and neuronal cell lines. Our results provide the first negative control in support of the hypothesis that adFNDI is caused solely by mutations in the AVP gene that alter amino acids residues crucial for folding and/or dimerization of the neurophysin II moiety of the AVP prohormone and its subsequent transport from the ER. We found that a mutation expected to impair proper folding of the neurophysin II moiety of the AVP prohormone by the substitution of histidine for tyrosine at position 2 in the hormone moiety (Y21H) (31, 32, 33) not only causes adFNDI (23) but also impairs transport of the mutant prohormone from the ER (Fig. 4Go) and subsequent processing to Y21H-AVP (Fig. 5Go) and neurophysin II (Figs. 1AGo and 3BGo). In addition, we found that another missense mutation, also predicting a substitution (P26L) of an amino acid residue not known and not reasonably expected (33, 34, 35) to be important for proper folding of the neurophysin II moiety of the AVP prohormone, not only fails to exert a dominant-negative effect or impair production of immunoreactive P26L-AVP in patients (22) but also has no effect on the intracellular trafficking of the mutant prohormone (Figs. 4Go, 1AGo, and 3CGo).

Our results concerning the cellular handling of the P26L prohormone are unique among previous findings in that this specific mutant prohormone is not retained to any detectable degree in the ER (Fig. 4Go), its intracellular trafficking and secretion is not prevented (Figs. 1AGo and 3CGo), but its processing into neurophysin II and hormone appears to be affected as indicated by the fact that the amounts of processed neurophysin II and processed immunoreactive P26L-AVP in the cell culture medium is reduced, compared with the amounts in the culture medium from cells expressing pcDNA3.1-WT (Figs. 1AGo and 5Go). The P26L substitution involves an amino acid residue in the C-terminal tail of the hormone moiety of the AVP prohormone, which has never been identified to be important for folding of the neurophysin II domain and which is oriented toward the solvent in the crystal structure of the oxytocin-neurophysin complex (35). As evidenced by the crystal structure and solution data on bound hormones (34), the C-terminal tails of the investigated hormones at best participate very weakly in binding to neurophysin. This makes it reasonable to propose that the apparently unaffected intracellular transport of the P26L prohormone observed in the present study occurs because the mutation has no or little effect on mutant prohormone folding and dimerization in the ER. If the intracellular transport of the mutated prohormone is efficient and without any major interference with the cellular integrity of the magnocellular neurons in individuals heterozygous for this mutation, it would allow unhindered secretion of sufficient amounts of AVP produced by the normal allele of the AVP gene to maintain the required antidiuretic function, thereby explaining their lack of DI (22). Likewise, it could explain the presence of the intact neurohypophysis in homozygous individuals as evidenced by the presence of the bright spot in one of the three affected family members as well as by their ability to secrete large amounts of immunoreactive P26L-AVP.

In the crystal structure of the neurophysin-oxytocin complex, the carbonyl oxygen of P26 forms a hydrogen bond with the backbone amide NH of G28 in the hormone (35), and conformational studies on synthetic pro-oxytocin-neurophysin precursors suggest that the presence of a ß-turn between P26 and G28 probably is important for the interaction with the active site of the prohormone convertase responsible for processing (36, 37, 38). Based on these structural data, it is tempting to speculate that the reduced amount of processed neurophysin II and processed immunoreactive P26L-AVP observed in the cell culture medium in the present study (Figs. 1AGo and 5Go) is due to a reduced efficiency of the enzymatic processing of the mutant prohormone into mature P26L-AVP and neurophysin II as a result of structural changes in the C-terminal tail of the hormone moiety induced by the P26L amino acid substitution. Such a reduced efficiency of the enzymatic processing of the mutant prohormone would imply an accumulation of precursors inside the cells. No such accumulation was detected in the present study. However, this could reflect either that the precursors somehow were rendered inaccessible or degraded by the cell or that they were not detected by the methodologies applied. Potential examples of the latter include restriction of labeling to 18 h, an inability of the confocal laser-scanning microscopic analysis to differentiate between precursors and processed products, or an inability of the RIA to detect incompletely processed hormone. It may seem striking that both Y21H-AVP and P26L-AVP levels are reduced, compared with WT in the cell culture model system when individuals homozygous for the mutation causing arFNDI remain to have the ability to secrete large amounts of immunoreactive P26L-AVP (22). However, it is actually not possible to decide from the clinical investigations of the affected individuals whether the apparent high levels of P26L-AVP reflect a high, normal, or reduced secretion capacity and whether the efficiency of the enzymatic processing of the mutant prohormone is affected because the normal hormone secretion capacity of their neurohypophysis is unknown. In any case, the clinical significance of a possible reduced efficiency of the enzymatic processing would probably be insignificant because it has been demonstrated that the mature P26L-AVP by itself is a weak agonist with decreased binding to the V2 receptor (22).

Our results concerning the cellular handling of the Y21H prohormone are generally consistent with previous findings (11, 12, 13, 14, 15, 16, 17, 18, 19, 20) in that it, compared with the WT, is largely retained in the ER and inefficiently processed into Y21H-AVP and neurophysin II. We have argued that the ER retention of the Y21H prohormone is caused by the failure of the altered prohormone to fold and dimerize properly in the ER (20). Although a small amount of Y21H prohormone apparently escapes ER retention and although the Y21H prohormone is subjected to intracellular degradation probably as a result of the action of the ER quality control of the cells recognizing and degrading incompletely folded and assembled protein (39, 40), it is clearly evident from our immunocytochemical observations that it accumulates in the ER. It can be speculated that this accumulation reflects a disturbed function of the ER as seen in, for example, Alzheimer’s and Parkinson’s disease (41), the ER-associated degradation of misfolded protein being affected in the former and the unfolded protein response being down-regulated in the latter. If the same things occur in patients with adFNDI, such cellular processes could either interfere with the secretion of WT prohormone or impair the viability of the AVP-producing cells, thereby explaining the delayed onset of the disease, its probable progressive development, and the dominant-negative effect exerted by the mutations.

In general, our data on the cellular handling of the P26L prohormone provide an important negative control in support of the hypothesis that adFNDI is caused solely by mutations in the AVP gene that alter amino acid residues important for folding and/or dimerization of the neurophysin II moiety of the AVP prohormone and its subsequent transport from the ER. Such a control could prove to have important value in future studies aiming at clarifying other important aspects concerning the pathogenesis of adFNDI, including the molecular mechanisms involved in ER retention, the role of protein degradation, and the cellular pathways eventually leading to degeneration of the neurons.


   Acknowledgments
 
The authors thank Jane Hagelskjær Knudsen for her skilled laboratory assistance.


   Footnotes
 
This work was supported by grants from the Karen Elise Jensen Fund, Novo Nordic Foundation, and University of Aarhus (Aarhus, Denmark).

Abbreviations: adFNDI, Autosomal dominant of FNDI; arFNDI, autosomal recessive familial neurohypophyseal diabetes insipidus; AVP, arginine vasopressin; DI, diabetes insipidus; Endo H, endoglycosidase H; ER, endoplasmic reticulum; FCS, fetal calf serum; irAVP, AVP immunoreactivity; WT, wild type.

Received October 17, 2003.

Accepted May 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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