Mutant Vasopressin Precursors That Cause Autosomal Dominant Neurohypophyseal Diabetes Insipidus Retain Dimerization and Impair the Secretion of Wild-type Proteins
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.9029
ISSN1083-351X
AutoresMika Ito, Richard N. Yu, J. Larry Jameson, Masafumi Ito,
Tópico(s)Ion Transport and Channel Regulation
ResumoAutosomal dominant familial neurohypophyseal diabetes insipidus is caused by mutations in the arginine vasopressin (AVP) gene. We demonstrated recently that mutant AVP precursors accumulate within the endoplasmic reticulum of neuronal cells, leading to cellular toxicity. In this study, the possibility that mutant AVP precursors interact with wild-type (WT) proteins to alter their processing and function was explored. WT and mutant precursors were epitope-tagged to allow them to be distinguished in transfected cells. An in vivo cross-linking reaction revealed homo- and heterodimer formation between WT and mutant precursors. Mutant precursors were also shown to impair intracellular trafficking of WT precursors from the endoplasmic reticulum to the Golgi apparatus. In addition to the cytotoxicity caused by mutant AVP precursors, the interaction between the WT and mutant precursors suggests that a dominant-negative mechanism may also contribute to the pathogenesis of familial neurohypophyseal diabetes insipidus. Autosomal dominant familial neurohypophyseal diabetes insipidus is caused by mutations in the arginine vasopressin (AVP) gene. We demonstrated recently that mutant AVP precursors accumulate within the endoplasmic reticulum of neuronal cells, leading to cellular toxicity. In this study, the possibility that mutant AVP precursors interact with wild-type (WT) proteins to alter their processing and function was explored. WT and mutant precursors were epitope-tagged to allow them to be distinguished in transfected cells. An in vivo cross-linking reaction revealed homo- and heterodimer formation between WT and mutant precursors. Mutant precursors were also shown to impair intracellular trafficking of WT precursors from the endoplasmic reticulum to the Golgi apparatus. In addition to the cytotoxicity caused by mutant AVP precursors, the interaction between the WT and mutant precursors suggests that a dominant-negative mechanism may also contribute to the pathogenesis of familial neurohypophyseal diabetes insipidus. Familial neurohypophyseal diabetes insipidus (FNDI) 1The abbreviations used are:FNDI, familial neurohypophyseal diabetes insipidus; AVP, arginine vasopressin; NP, neurophysin; WT, wild-type; HA, hemagglutinin; BFA, brefeldin A; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; Endo H, endoglycosidase H; DSS, disuccinimidyl suberate.1The abbreviations used are:FNDI, familial neurohypophyseal diabetes insipidus; AVP, arginine vasopressin; NP, neurophysin; WT, wild-type; HA, hemagglutinin; BFA, brefeldin A; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; Endo H, endoglycosidase H; DSS, disuccinimidyl suberate. (1Baylis P.H. DeGroot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 406-420Google Scholar) is caused by a deficiency of arginine vasopressin (AVP), a hormone that controls serum osmolality by altering renal free water clearance (1Baylis P.H. DeGroot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 406-420Google Scholar). Diabetes insipidus is transmitted as an autosomal dominant trait in these families. A large number of distinct mutations have been found in the AVP gene (2Ito M. Mori Y. Oiso Y. Saito H. J. Clin. Invest. 1991; 87: 725-728Crossref PubMed Scopus (119) Google Scholar, 3Bahnsen U. Oosting P. Swaab D.F. Nahke P. Richter D. Schmale H. EMBO J. 1992; 11: 19-23Crossref PubMed Scopus (94) Google Scholar, 4Ito M. Oiso Y. Murase T. Kondo K. Saito H. Chinzei T. Racchi M. Lively M.O. J. Clin. Invest. 1993; 91: 2565-2571Crossref PubMed Scopus (94) Google Scholar, 5Yuasa H. Ito M. Nagasaki H. Oiso Y. Miyamoto S. Sasaki N. Saito H. J. Clin. Endocrinol. Metab. 1993; 77: 600-604Crossref PubMed Scopus (0) Google Scholar, 6Krishnamani M.R. Phillips J.A.I. Copeland K.C. J. Clin. Endocrinol. Metab. 1993; 77: 596-598Crossref PubMed Scopus (0) Google Scholar, 7McLeod J.F. Kovacs L. Gaskill M.B. Rittig S. Bradley G.S. Robertson G.L. J. Clin. Endocrinol. Metab. 1993; 77: 599ACrossref PubMed Scopus (91) Google Scholar, 8Repaske D.R. Browning J.E. J. Clin. Endocrinol. Metab. 1994; 79: 421-427Crossref PubMed Google Scholar, 9Nagasaki H. Ito M. Yuasa H. Saito H. Fukase M. Hamada K. Ishikawa E. Katakami H. Oiso Y. J. Clin. Endocrinol. Metab. 1995; 80: 1352-1356PubMed Google Scholar, 10Rauch F. Lenzner C. Nurnberg P. Frommel C. Vetter U. Clin. Endocrinol. 1996; 44: 45-51Crossref PubMed Scopus (32) Google Scholar, 11Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregerse N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google Scholar, 12Rutishauser J. Boni-Schnetzler M. Boni J. Wichmann W. Huisman T. Vallotton M.B. Froesch E.R. J. Clin. Endocrinol. Metab. 1996; 81: 192-198PubMed Google Scholar, 13Ueta Y. Taniguchi S. Yoshida A. Murakami I. Mitani Y. Hisatome I. Manabe I. Sato R. Tsuboi M. Ohtahara A. Nanba E. Shigemasa C. J. Clin. Endocrinol. Metab. 1996; 81: 1787-1790PubMed Google Scholar, 14Heppner C. Kotzka J. Bullmann C. Krone W. Muller-Wieland D. J. Clin. Endocrinol. Metab. 1998; 83: 693-696Crossref PubMed Scopus (37) Google Scholar, 15Calvo B. Bilbao J.R. Urrutia I. Eizaguirre J. Gaztambide S. Castano L. J. Clin. Endocrinol. Metab. 1998; 83: 995-997Crossref PubMed Scopus (35) Google Scholar). The AVP gene encodes polypeptide precursors consisting of a signal peptide, AVP, neurophysin (NP), and glycoprotein domains (16Sausville E. Carney D. Battey J. J. Biol. Chem. 1985; 260: 10236-10241Abstract Full Text PDF PubMed Google Scholar). Prepro-AVP is synthesized in the magnocellular neurons of the hypothalamus and is converted to pro-AVP by the removal of the signal peptide. Pro-AVP undergoes several post-translational processing steps, including the addition of carbohydrate side chains and proteolytic cleavage to yield AVP, NP, and the glycoprotein. These products are stored within neurosecretory vesicles in the axonal terminals of the posterior pituitary gland and are secreted into the blood in response to osmotic stimuli (17Brownstein M.J. Russel J.T. Gainer H. Science. 1980; 207: 373-378Crossref PubMed Scopus (627) Google Scholar). Most of the mutations in individuals with FNDI have been found within the signal peptide and the NP domains (18Hansen L.K. Rittig S. Robertson G.L. Trends Endocrinol. Metab. 1997; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). A substitution of Thr for Ala at the carboxyl terminus of the signal peptide (A(−1)T) is the most commonly found mutation, and it has been identified in various ethnic groups, suggesting independent mutational events. Mutations within the NP domain include amino acid substitutions, a single amino acid deletion, and premature protein termination. Despite the presence of a normal allele, FNDI patients develop diabetes insipidus, although the symptoms are not usually manifest until several months or years after birth. These features have raised questions concerning the molecular pathogenesis of the disorder. A limited number of autopsy studies have demonstrated a paucity of AVP-producing neurons in the hypothalamus of patients with FNDI (19Braverman L.E. Mancini J.P. McGoldrick D.M. Ann. Intern. Med. 1965; 63: 503-508Crossref PubMed Scopus (96) Google Scholar, 20Green J.R. Buchan G.C. Alvord E.C. Swanson A.G. Brain. 1967; 90: 707-714Crossref PubMed Scopus (83) Google Scholar, 21Nagai I. Li C.H. Hsieh S.M. Kizaki T. Urano Y. Acta Endocrinol. 1984; 105: 318-323Crossref PubMed Scopus (43) Google Scholar, 22Bergeron C. Kovacs K. Ezrin C. Mizzen C. Acta Neuropathol. 1991; 81: 345-348Crossref PubMed Scopus (83) Google Scholar). Consistent with these pathologic findings, magnetic resonance imaging has revealed an absence of the bright spot that characterizes the posterior pituitary gland in a subset of patients with FNDI (12Rutishauser J. Boni-Schnetzler M. Boni J. Wichmann W. Huisman T. Vallotton M.B. Froesch E.R. J. Clin. Endocrinol. Metab. 1996; 81: 192-198PubMed Google Scholar). Based on these observations, it has been postulated that mutant AVP precursors might be cytotoxic to AVP-producing neurons. In a previous study (4Ito M. Oiso Y. Murase T. Kondo K. Saito H. Chinzei T. Racchi M. Lively M.O. J. Clin. Invest. 1993; 91: 2565-2571Crossref PubMed Scopus (94) Google Scholar), the A(−1)T mutation was shown to cause inefficient cleavage of the signal peptide, giving rise to aberrant precursors that were glycosylated, but not cleaved, by signal peptidase. This finding raised the possibility that the aberrant precursors might accumulate and lead to cellular toxicity. In support of this idea, the expression of several different FNDI mutants was shown to impair the intracellular trafficking of mutant AVP precursors and the viability of neuroblastoma cells (23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). The cytotoxicity of mutant AVP precursors may be sufficient to account for the autosomal dominant mode of inheritance of FNDI. However, in some autosomal dominant diseases, the mutant protein exerts dominant-negative activity to alter the function of the normal allele (24Herskowitz I. Nature. 1987; 329: 219-222Crossref PubMed Scopus (859) Google Scholar). AVP precursors have been shown to physically interact with each other in vitro (25Kanmera T. Chaiken I.M. J. Biol. Chem. 1985; 260: 8474-8482Abstract Full Text PDF PubMed Google Scholar). It is therefore possible that mutant AVP precursors could form heterodimers with wild-type (WT) protein products to alter their function and contribute to the pathogenesis of FNDI. In this study, we examined the physical and functional interactions between WT and mutant AVP precursors by expressing epitope-tagged precursors in cultured cells. Expression vectors for the WT and mutant (G57S (2Ito M. Mori Y. Oiso Y. Saito H. J. Clin. Invest. 1991; 87: 725-728Crossref PubMed Scopus (119) Google Scholar), A(−1)T (4Ito M. Oiso Y. Murase T. Kondo K. Saito H. Chinzei T. Racchi M. Lively M.O. J. Clin. Invest. 1993; 91: 2565-2571Crossref PubMed Scopus (94) Google Scholar), ΔE47 (5Yuasa H. Ito M. Nagasaki H. Oiso Y. Miyamoto S. Sasaki N. Saito H. J. Clin. Endocrinol. Metab. 1993; 77: 600-604Crossref PubMed Scopus (0) Google Scholar), and C67X (9Nagasaki H. Ito M. Yuasa H. Saito H. Fukase M. Hamada K. Ishikawa E. Katakami H. Oiso Y. J. Clin. Endocrinol. Metab. 1995; 80: 1352-1356PubMed Google Scholar)) AVP precursors (Fig. 1 A) have been described previously (23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). For epitope tagging (Fig. 1 B), restriction sites for ClaI, SpeI, andXbaI (ATCGAT ACTAGT TCTAGA) were introduced by polymerase chain reaction immediately after the last codon of the glycoprotein domain (WT, G57S, A(−1)T, and ΔE47) or the 66th codon of the NP domain (C67X), and the resulting vectors were digested withClaI and XbaI. Annealed oligonucleotides containing the Myc-His tag or the influenza hemagglutinin (HA) tag, along with sequence overhangs for the ClaI andXbaI sites, were ligated into the same restriction sites in the plasmid vectors (Fig. 1 B). The Myc-His tag contains the c-Myc epitope (EQKLISEEDL), the intervening amino acid sequence (NSAVD), and polyhistidine sequence (His6) from the pcDNA3.1MycHis expression vector (Invitrogen, San Diego, CA). The His6 sequence is added to allow protein purification. The amino acid sequence of the HA tag is YPYDVPDYA. After polymerase chain reaction and subcloning, the entire cDNA sequence was verified by the dideoxy-mediated chain termination method (26Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52232) Google Scholar). All the cDNAs were introduced into the pRc/RSV vector (Invitrogen). The vector without a cDNA insert was used as a control in some experiments. Human embryonic kidney tsa 201 cells (27Margolskee R.F. McHendry-Rinde B. Horn R. BioTechniques. 1993; 15: 906-911PubMed Google Scholar) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2atmosphere at 37 °C. Cells were transfected by the calcium phosphate method as described previously (28Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-487Crossref PubMed Scopus (6460) Google Scholar). In precursor interaction assays (see below), cells were treated with 10 μg/ml brefeldin A (BFA) (Sigma) for 12 h to inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus (29Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar). Continuous metabolic labeling and immunoprecipitation were performed as described previously (23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). Briefly, transiently transfected cells were labeled for 12 h in Dulbecco's modified Eagle's medium containing 100 μCi of Expre35S35S protein labeling mixture (DuPont). Cell extracts and culture medium were subjected to immunoprecipitation using polyclonal anti-AVP or anti-NP antibodies (ICN, Costa Mesa, CA). Immunoprecipitates were separated by 16.5% SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. For pulse-chase analyses, transfected cells were labeled for 15 min with 100 μCi of Expre35S35S protein labeling mixture in 0.5 ml of methionine- and cysteine-free Dulbecco's modified Eagle's medium and chased using 1 ml of complete Dulbecco's modified Eagle's medium containing 100 μg/ml cycloheximide (Sigma). Cycloheximide was added to completely inhibit further synthesis of labeled precursors after the pulse labeling. In some experiments, immunoprecipitates were treated with endoglycosidase H (Endo H) (New England Biolabs Inc., Beverly, MA) according to the instructions of the manufacturer. Densitometric analyses were performed using a GS-700 imaging densitometer (Bio-Rad). Quantitative analyses were performed with film exposures in the linear range. Cells transfected with expression vectors for precursors, with or without the Myc-His epitope tag, were labeled for 12 h in the presence of 10 μg/ml BFA. After labeling, cells were lysed in buffer A (20 mm Tris (pH 8.0), 150 mm NaCl, 1% Triton X-100, 5 mmimidazole, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). Cell extracts were incubated with the Talon metal affinity resin (CLONTECH, Palo Alto, CA) in the presence of 10 mm imidazole for 30 min at 4 °C with gentle rocking. After extensive washing in buffer A containing 15 mmimidazole, bound proteins were eluted from the resin by boiling in SDS-PAGE sample buffer containing dithiothreitol and subjected to 16.5% SDS-PAGE followed by autoradiography. For the in vitro precursor interaction assay, WT precursors with and without the Myc-His tag were translated in vitro using the TNT reticulocyte lysate system (Promega, Madison, WI) in the presence of [35S]methionine (DuPont) and canine microsomal membranes (Promega). The [35S]methionine-labeled proteins were subjected to the binding reaction with metal affinity resin as described above. Extensive washing and the inclusion of imidazole throughout the assay are necessary to reduce nonspecific protein interactions. Under these conditions, <10% of the total input protein is typically bound to the affinity resin (30Ito M. Yu R. Jameson J.L. Mol. Cell. Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (387) Google Scholar). A non-cleavable cross-linker, disuccinimidyl suberate (DSS) (Pierce), was dissolved in dimethyl sulfoxide at a concentration of 100 mm. For the detection of homodimers, cells expressing precursors with the HA tag were collected in phosphate-buffered saline and then incubated in either phosphate-buffered saline containing Me2SO (1:100 dilution) or phosphate-buffered saline containing 1 mm DSS for 30 min at room temperature. Immediately after performing the cross-linking reaction, cells were lysed in a buffer containing 20 mm Hepes (pH 7.9), 420 mm NaCl, 20% glycerol, 1.5 mmMgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A. Whole cell extracts were subjected to 15% reducing SDS-PAGE followed by electrotransfer to polyvinylidene difluoride membranes (Boehringer Mannheim). Membranes were probed with horseradish peroxidase-conjugated anti-HA antibody (Boehringer Mannheim) according to the instructions of the manufacturer. Subsequently, proteins were detected using the enhanced chemiluminescence detection system (Boehringer Mannheim). For the detection of heterodimers as well as homodimers, cells expressing precursors with the Myc-His tag and those with the HA tag were subjected to an in vivo cross-linking reaction as described above. After the reaction, cells were lysed in buffer A, and cell extracts were incubated with the metal affinity resin as described above for the precursor interaction assay. Bound proteins were eluted from the resin and separated by 15% SDS-PAGE under reducing conditions. After Western blot transfer, membranes were probed with the anti-HA antibody. Human embryonic kidney tsa 201 cells were transiently transfected with an empty vector or with expression vectors for WT AVP precursors with or without the Myc-His epitope tag to allow studies of precursor expression and processing. After continuous metabolic labeling, cell extracts and medium were harvested and subjected to immunoprecipitation. A 14-kDa protein (Fig.2 A, lanes 3,5, 9, and 11) was detected in cells transfected with an empty vector (lanes 1 and 7), suggesting that this band is nonspecific. Using the anti-NP antibody, a similar series of precursors were identified, independent of the presence of the Myc-His epitope tag. The WT AVP precursor was 21 kDa (lane 3), whereas the intracellular precursor containing the Myc-His epitope tag was 25 kDa (lane 5). The expression levels of the 21- and 25-kDa precursors for the WT and Myc-His-tagged AVP precursors were similar. The intracellular precursors consisted of doublets (lanes 3 and 5), presumably reflecting different states of glycosylation. In the medium, the 22-kDa (WT) (lane 4) and 26-kDa (Myc-His) (lane 6) precursors as well as a 12-kDa protein were detected. The sizes of the precursors in the medium were increased relative to those in the cell extracts, reflecting the addition of carbohydrate moieties prior to secretion (Fig. 2 B). Each of the precursor forms and the 12-kDa protein were also immunoprecipitated with the anti-AVP antibody (Fig.2 A, lanes 9–12), indicating that they contain both the AVP and NP domains. The 12-kDa protein corresponds to the intermediate form observed in neuro2A cells (Fig. 2 B) (23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). These experiments demonstrate that the sizes of the processed AVP precursor products in this cell line and indicate that the presence of the Myc-His epitope tag does not alter the level or the efficiency of precursor processing or secretion into the medium. Digestion of the immunoprecipitated proteins with Endo H (Fig.3 A) demonstrated that the WT intracellular precursors (21 and 25 kDa) (lanes 1 and11) were sensitive to Endo H, yielding 17- and 21-kDa digested products (lanes 21 and 31). In contrast, the products in the medium (lanes 2 and 12) were resistant to Endo H digestion (lanes 22 and 32). The Endo H resistance of the secreted precursors is consistent with their increased molecular size, likely reflecting glycosylation within the Golgi apparatus. Little or no Endo H-resistant precursors were detected within cells (lanes 21 and 31), suggesting that AVP precursors are rapidly exported into the medium or undergo further processing once they are glycosylated within the Golgi apparatus (Fig. 2 B). The expression and processing of the G57S, A(−1)T, ΔE47, and C67X mutant AVP precursors were examined in parallel with the WT precursors described above. Under conditions of continuous labeling (Fig. 3 A), the mutant AVP precursors with and without the Myc-His tag were readily detected within cells (lanes 1, 3, 5,7, and 9 and lanes 11, 13,15, 17, and 19). In fact, in most cases, the level of mutant precursor expression was greater than that of WT precursor expression (lanes 1 and 11). The migration of the G57S precursors (lanes 3 and 13) was indistinguishable from that of the WT precursors, whereas the A(−1)T (lanes 5 and 15) and ΔE47 (lanes 7 and 17) precursors migrated differently. The slower migration of the A(−1)T mutant likely reflects the formation of aberrant precursors that are glycosylated, but not cleaved by signal peptidase (Fig. 2 B) (4Ito M. Oiso Y. Murase T. Kondo K. Saito H. Chinzei T. Racchi M. Lively M.O. J. Clin. Invest. 1993; 91: 2565-2571Crossref PubMed Scopus (94) Google Scholar, 23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). The slightly faster migration of the ΔE47 mutant may reflect the deletion of a single amino acid. The C67X mutant, with and without the Myc-His tag (14 and 10 kDa) (Fig. 3 A, lanes 9 and 19), was also expressed well within cells. In the medium, 22- and 26-kDa precursors as well as 12-kDa intermediate forms were detected for the WT, G57S, and A(−1)T mutants (lanes 2, 4, and6 and lanes 12, 14, and16). The sizes of the secreted WT and A(−1)T mutant precursors in the medium were the same because their products are identical once the signal peptide is removed. The amount of the G57S and A(−1)T proteins detected in the medium was reduced compared with that of the WT protein. Little or no precursors were detectable in the medium for the Δ47E and C67X mutants (lanes 8and 10 and lanes 18 and 20). The effects of Endo H treatment were similar for the mutant and WT precursors (Fig. 3 A). Digestion of intracellular G57S, A(−1)T, and ΔE47 AVP precursor proteins with and without the Myc-His tag (lanes 3, 5, and 7 and lanes 13, 15, and 17) gave rise to 17- and 21-kDa proteins (lanes 23, 25, and 27 andlanes 33, 35, and 37). The G57S and A(−1)T precursors in the medium (lanes 4 and 6and lanes 14 and 16) were resistant to Endo H digestion (lanes 24 and 26 and lanes 34 and 36). No Endo H-resistant precursors were detected within the cells, indicating that like the WT precursors, most of the mutant precursors are readily secreted into the medium after glycosylation within the Golgi apparatus. As expected, Endo H had no effect on the C67X mutant precursors (lanes 9 and19 and lanes 29 and 39). Pulse-chase analyses were performed to further evaluate the kinetics of precursor processing and secretion. AVP precursors recovered in cells and in the medium 1 h after the pulse labeling were normalized to the amount of labeled products present after labeling (Fig.3 B, left panel). The recovery of WT precursors in the medium was 31%, whereas that of the mutants ranged between 0 and 7%, indicating reduced secretion of mutant precursors. The total recovery of WT precursors in cells and the medium was 71%, whereas the recovery of the C67X mutant precursor was only 35%. In the case of other mutant precursors, the recovery was between 50 and 64%. These results suggest that intracellular degradation is involved in the inefficient secretion of mutant precursors. Pulse-chase analyses also revealed that mutant precursors were still retained within cells 8 h after pulse labeling (Fig.3 B, right panel). By comparison, most of the WT precursors were secreted into the medium by 8 h, indicating that the mutant AVP precursors are secreted into the medium less efficiently than the WT precursors. Because precursors appear to be rapidly secreted into the medium after glycosylation (Fig. 3 A), these findings suggest that the G57S, A(−1)T, and ΔE47 mutant precursors are not transported from the ER to the Golgi apparatus as effectively as the WT precursors. The levels of the C67Xmutant were relatively low due to intracellular degradation (see above), but it, too, appears to be retained within the intracellular pool, consistent with previous studies in which ER retention of the C67X precursors was demonstrated using immunofluorescence staining in neuro2A cells (23Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (152) Google Scholar). Taken together, these results show that both intracellular degradation and retention are responsible for the reduced secretion of mutant precursors. AVP precursors were previously shown to interact with each other in vitro using synthetic peptides and immobilized NP (25Kanmera T. Chaiken I.M. J. Biol. Chem. 1985; 260: 8474-8482Abstract Full Text PDF PubMed Google Scholar). We used a protein "pull-down" assay to assess interactions among the precursor proteins. Cells were treated with BFA, which blocks protein transport from the ER to the Golgi apparatus, to allow a similar degree of retention of both mutant and WT AVP precursors. Treatment of cells with BFA completely eliminated the release of precursors and intermediate forms into the medium, resulting in a comparable degree of WT and mutant precursor expression (data not shown). Cells expressing precursors with and without the Myc-His tag were labeled in the presence of BFA prior to lysis. Cell extracts were incubated with metal affinity resin to bind AVP precursors containing the Myc-His tag to the resin. After extensive washing, bound proteins were separated on 16.5% SDS-polyacrylamide gels followed by autoradiography (Fig.4 A). AVP precursors with the Myc-His tag were retained by the metal affinity resin (lane 1), but those without the Myc-His tag were not retained (lane 2), demonstrating a specific interaction of the His6-tagged precursors with the resin. WT and mutant precursors with the Myc-His tag interacted with their respective precursors without the tag (Fig. 4 A, lanes 3–7), as reflected by the fact that the precursors without the Myc-His tag were also retained by the affinity column. These results suggest that WT and mutant precursors form homodimers. WT precursors with the Myc-His tag also interacted with the G57S, A(−1)T, ΔE47, and C67X mutant precursors (lanes 8–11). And in the reverse format, the G57S, A(−1)T, ΔE47, and C67Xmutant precursors with the Myc-His tag interacted with the WT precursors (lanes 12–15). These results indicate that the WT and mutant precursors form both homo- and heterodimers. Precursor proteins were also labeled during in vitrotranslation to provide an estimate of the fraction of input proteins that interact with the metal affinity resin in this assay (Fig.4 B). WT precursors with and without the Myc-His tag were translated in vitro in the presence of microsomal membranes to produce prohormones (4Ito M. Oiso Y. Murase T. Kondo K. Saito H. Chinzei T. Racchi M. Lively M.O. J. Clin. Invest. 1993; 91: 2565-2571Crossref PubMed Scopus (94) Google Scholar). WT precursors with the Myc-His tag (25 kDa) (lane 2) interacted with metal affinity resin (lane 4). WT precursors (21 kDa) (lane 1) without the tag did not interact with the resin (lane 3), but were retained in the presence of WT precursors with the Myc-His tag (lane 5). In comparison with the input proteins (lanes 2and 4), ∼8% of the WT precursors with the Myc-His tag were bound, which is similar to the interactions of previously characterized protein dimers in this type of assay (30Ito M. Yu R. Jameson J.L. Mol. Cell. Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (387) Google Scholar). Approximately 7% of the total input of WT precursors without the tag was associated with WT precursors with the Myc-His tag (lanes 1and 5), suggesting that the majority of WT precursors without the tag interact with WT precursors with the epitope tag. Cross-linking studies were used to further assess the formation of AVP precursor homodimers within the cellular environment. WT or mutant precursors with the HA tag were expressed, and the cells were subjected to an in vivo cross-linking reaction with DSS prior to lysis. After Western blot transfer, membranes were probed with an anti-HA antibody (Fig. 5). The monomeric form of the WT, G57S, A(−1)T, and ΔE47 precursors was 23 kDa, whereas the C67X monomer was 12 kDa. In the absence of DSS treatment, homodimerization of the G57S and ΔE47 precursors (46 kDa) was detected (lanes 3 and 7), but little or no homodimerization was seen with the WT, A(−1)T, and C67X (24 kDa) precursors (lanes 1,5, and 9). After treatment with DSS, all of the WT and mutant precursors formed homodimers (lanes 2, 4, 6, 8, and10). The ratio of dimer to monomer for the C67Xmutant was greater than that for the WT, G57S, A(−1)T, and ΔE47 precursors. Cells coexpressing WT and mutant precursors with the HA tag and the Myc-His tag were subjected to the cross-linking reaction with DSS prior to lysis. Cell extracts were prepared and incubated with the metal affinity resin to isolate Myc-His-tagged complexes. After washing, bound proteins were subjected to Western blot transfer, and the membranes were probed with the anti-HA antibody (Fig.6). As a control, WT precursors with the Myc-His tag were not recognized by the anti-HA antibody (lane 1). WT precursors with the HA tag were not detected in the absence of precursors with the Myc-His tag (lane 2), indicating that the precursors with the HA tag were not retained by the affinity resin. However, WT precursors with the HA tag were detected when coexpressed with WT precursors containing the Myc-His tag (lane 3). The detection of 48-kDa proteins by the anti-HA antibody indicates the formation of mixed complexes consisting of WT precursors with the Myc-His tag (25 kDa) and those with the HA tag (23 kDa) (lane 3). Similarly, homodimerization of G57S, A(−1)T, and ΔE47 precursors was demonstrated by the detection of complexes con
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