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Endocrinology 145(4):1961–1971 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-1472
Furin and Prohormone Convertase 1/3 Are Major Convertases in the Processing of Mouse Pro-Growth Hormone-Releasing Hormone ARUNANGSU DEY, CHRISTINA NORRBOM, XIAORONG ZHU, JEFFREY STEIN, CHUNLING ZHANG, KAZUYA UEDA, AND DONALD F. STEINER From the Department of Biochemistry and Molecular Biology (A.D., C.N., X.Z., J.S., D.F.S.) and the Howard Hughes Medical Institute (C.Z., K.U., D.F.S.), University of Chicago, Chicago, Illinois 60637 We investigated the proteolytic processing of mouse proGHRH [84 amino acids (aa)] by furin, PC1/3, PC2, and PC5/6A. We created six point mutations in the N- and C-terminal cleavage sites, RXXR2 and RXRXXR2, respectively. The following results were obtained after transient transfection/cotransfection and metabolic pulse-chase labeling studies in several neuroendocrine cells. 1) Furin was the most efficient convertase in cleaving the N-terminal RXXR/RXRR site to generate intermediate I, 12– 84aa, whereas PC1/3 was the most potent in processing the C-terminal RXRXXR site to yield mature GHRH, 12–53aa. 2) Both PC1/3 and PC5/6A also processed the N-terminal site but less efficiently than furin. 3) PC2 was much
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RO-GHRH IS PROCESSED in the hypothalamus to generate bioactive/mature GHRH that is released into the hypothalamic-pituitary portal vessels and ultimately carried to the pituitary gland to control the synthesis and secretion of GH from the anterior pituitary somatotrophs (1). After the cloning and characterization of human and rat pro-GHRH (2– 4), mouse pro-GHRH was cloned and shown to have an open reading frame of 103 amino acids (aa) including a signal sequence of 19 aa (5, 6) (see Fig. 1). Mouse pro-GHRH shows 68 and 62% amino acid identity to the rat and human precursors, respectively. It is noteworthy that the C-terminal prosegment of mouse pro-GHRH has greater homology with rat than human, especially at the beginning of exon 5. Among other noteworthy differences are: 1) human GHRH has an amidated C terminus due to the presence of Gly at the P2 position in the C-terminal cleavage site, compared with the nonamidated mouse and rat GHRH, and 2) human GHRH has Tyr at its N terminus relative to His in mouse and rat. In mouse, the N-terminal processing site for generation of mature GHRH, RXXR2, differs from the N-terminal cleavage site of rat and human, RXRR2, whereas the C-terminal RXRXXR2 processing site is the same in all three. These are typical recognition sequences for prohormone convertase (PC) mediated processing. GHRH-containing neurons are Abbreviations: aa, Amino acid; ECFP, enhanced cyan fluorescence protein; EGFP, enhanced green fluorescence protein; FBS, fetal bovine serum; IP, immunoprecipitation; ISG, immature secretory granule; PC, prohormone convertase; ␣1-PDX, ␣1-antitrypsin Portland; TGN, transGolgi network. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
weaker in cleaving the C-terminal site relative to PC1/3 to generate mature GHRH. 4) The Q10R mutant was significantly more susceptible to furin cleavage at the N-terminal site than the wild-type pro-GHRH. And 5) the N- and C-terminal P1 Arg residues, R11 and R54, respectively, were essential for mature GHRH production. We also showed localization of the GHRH immunoreactive peptides in Golgi and secretory granules in neuroendocrine cells by an immunofluorescence assay. We conclude that the efficient production of mature GHRH from pro-GHRH is a stepwise process mediated predominantly by furin at the N-terminal cleavage site followed by PC1/3 at the C terminus. (Endocrinology 145: 1961–1971, 2004)
shown to localize primarily in the arcuate nucleus of the hypothalamus, and only a few thousand such neurons have been identified (7, 8), rendering studies of the processing of pro-GHRH in vivo difficult. After the discovery of the yeast convertase, Kex2 (9, 10), seven mammalian basic residue cleaving homologs have been identified during the past decade of which two members, PC1/3 and PC2, are abundantly expressed in neuroendocrine tissues (11–18), and among the rest, furin, PC5/6A, PC5/6B, PACE4, and PC7 are broadly distributed (19 –33), whereas PC4 is expressed in testicular and ovarian tissues (34 –37). Studies have shown that PC5/6A is present in secretory granules of neuroendocrine cells similar to PC1/3 and PC2 (38 – 43), whereas furin, apart from its trans-Golgi network (TGN)/endosomal localization, can also be sorted transiently into immature secretory granules (ISGs) of the regulated secretory pathway in neuroendocrine cells (44 – 46). Whereas the roles of PC1/3 and PC2 in processing various prohormones and proneuropeptides are well documented including in animal models (47– 62), several studies have shown the abilities of PC5/6A and furin to process a growing number of prohormones and proneuropeptides such as pro-melanin-concentrating hormone (56), proneurotensin/neuromedin (63, 64), pro-cholecystokinin (65), pro-PTH (66), pro-TRH (67), proinsulin (68), chromogranin A (69), and 7B2 (70) in various systems having regulated and/or constitutive secretory pathway. Recently a PC1/3 inhibitory protein pro-SAAS, distributed mostly in neuroendocrine tissues, has been shown to be cleaved by furin both in vitro and in neuroendocrine cells (71, 72). In the current studies using several different neuroendo-
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FIG. 1. Schematic diagram of wild-type mouse pro-GHRH with the two terminal PC cleavage sites and the six point mutations made therein. The N-terminal processing site is represented by R(Arg) M(Met) Q(Gln) R(Arg) and the C-terminal cleavage site is indicated by R(Arg) A(Ala) R(Arg) L(Leu) S(Ser) R(Arg). Each number refers to a position of a specific amino acid in either the N or C terminus of the pro-GHRH sequence. Each small arrow denotes a specific point mutation, and the two big arrows indicate the N- and C-terminal cleavage sites on pro-GHRH, respectively.
crine cells, we performed detailed analyses to understand the roles of the two classical neuroendocrine convertases, PC1/3 and PC2, as well as the two broadly distributed convertases, furin and PC5/6A, in processing mouse pro-GHRH. Materials and Methods Sources The mouse pro-GHRH (84 aa) expression plasmid was a kind gift from Dr. Kelley E. Mayo (Northwestern University, Evanston, IL). The two rabbit polyclonal antibodies against the mouse GHRH, 4398 and 7802, and the purified rat GHRH peptide were kind gifts from Dr. Lawrence A. Frohman (University of Illinois, Chicago, IL). The furin inhibitor, ␣1-antitrypsin Portland (␣1-PDX), and recombinant human furin expression plasmid were kind gifts from Dr. Gary Thomas (Vollum Institute, Oregon Health Sciences University, Portland, OR). The expression plasmids for mouse PC2 and PC1/3 were generated in our laboratory. Neuro-2A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). RPE.40 cells were kindly provided by Dr. Thomas Moehring (University of Vermont, Burlington, VT). All the cell culture and PCR reagents were purchased from Invitrogen/Life Technologies (Carlsbad, CA). The transfection reagent, Effectene, and the endotoxin-free plasmid DNA purification kit were from Qiagen (Valencia, CA).
Construction of the recombinant plasmids expressing the Nand C-terminal point mutants of mouse pro-GHRH Altogether six point mutants, three at the N-terminal RXXR site and three at the C-terminal RXRXXR site, were constructed (Fig. 1). For the N-terminal site, Arg11 (R11) and Arg8 (R8) at positions P1 and P4, respectively, were individually changed to Ala (A), and Gln10 (Q10) at position P2 was substituted by Arg (R) using the overlap extension PCR method. After PCR, both the R11A and R8A mutant cDNAs were ligated at the restriction sites HindIII (5⬘) and EcoRI (3⬘) in a mammalian expression vector, pcDNA3.1(⫹) (Invitrogen), whereas the Q10R mutant was ligated at HindIII (5⬘) and XbaI (3⬘) in the same expression vector. The oligonucleotide primers used for the overlap extension PCR were the following: 1) The upstream and downstream primers for both the R11A and R8A were (F) CCC AAG CTT CAG GAG TGA AGG ATG CTG CTC TGG GTG and (R) GGC GAA TTC TCG GGG GGC TCA AGC GTC CGC TGA AGG; and 2) for the Q10R, the upstream (F) primer was the same as above and the downstream primer was (R) GGC TCT AGA TCG GGG GGC TCA AGC GTC CGC TGA AGG. The internal primers were the following: 1) R11A, (R) GAT GGC ATC TAC GTG GGC CTG CAT CCT GAA GGG and (F) CCC TTC AGG ATG CAG GCC CAC GTA GAT GCC ATC; 2) R8A, (R) TAC GTG TCG CTG CAT GGC GAA GGG AGG TGA GGG and (F) CCC TCA CCT CCC TTC GCC ATG CAG CGA CAC GTA; and 3) Q10R, (R) GGC ATC TAC GTG TCG TCG CAT CCT GAA GGG AGG and (F) CCT CCC TTC AGG ATG CGA CGA CAC GTA GAT
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GCC. The thermocycle used for the overlap extension PCR was one hold at 94 C for 3 min followed by 25 cycles at 94 C for 30 sec, 55 C for 1 min, 72 C for 2 min, and finally one hold at 72 C for 5 min. For the C-terminal mutants, each of Arg54 (R54), Arg51 (R51), and Arg49 (R49) at positions P1, P4, and P6, respectively, was changed to Ala (A) by using the site-directed mutagenesis kit from Stratagene (La Jolla, CA) and the pcDNA3.1(⫹) vector having the wild-type pro-GHRH cDNA insert as the template DNA. The oligonucleotide primers used for the site directed mutagenesis were the following: 1) R54A, (F) AGG GCC AGG CTC AGC GCC CAG GAA GAC AGC ATG and (R) CAT GCT GTC TTC CTG GGC GCT GAG CCT GGC CCT; 2) R51A, (F) CAG GAA CAA AGG GCC GCC CTC AGC CGC CAG G and (R) C CTG GCG GCT GAG GGC GGC CCT TTG TTC CTG; and 3) R49A, (F) GG ATC CAG GAA CAA GCC GCC AGG CTC AGC CGC and (R) GCG GCT GAG CCT GGC GGC TTG TTC CTG GAT CC. The PCR temperature used for the site-directed mutagenesis was one hold at 95 C for 30 sec followed by 16 cycles at 95 C for 30 sec, 55 C for 1 min, and 68 C for 12 min. All of the mutant constructs were verified by sequencing using the PE Biosystems kit (Warrington, UK).
Cell culture, metabolic labeling, and immunoprecipitation (IP) GH4C1 cells were cultured in F10 (Ham) with 10% fetal bovine serum (FBS), RPE.40 cells were maintained in F12 (Ham) with 5% FBS, and Neuro-2A cells were cultured in Eagle’s MEM with 10% FBS. ␣TC1– 6, TC-3, and AtT 20 cells were grown in DMEM (high glucose) with 10% FBS, and LoVo cells were cultured in F12 with 20% FBS and 4 mm glutamine. Transient transfection/cotransfection of cells with either the wildtype or each of the mutant pro-GHRH expression plasmids in absence or presence of different prohormone convertase expression plasmids were done according to the Qiagen’s protocol. Pulse-chase labeling of transfected cells with [35S]-Met followed by IP of the radioactively labeled peptides with the rabbit antimouse GHRH (no. 4398) were performed as exactly described before (47). In the current studies, we used the protein-A-agarose beads (Roche Diagnostics, Indianapolis, IN) for all IP reactions. Immunoprecipitated peptides were finally analyzed in tricine/SDS-PAGE (73) under the reducing conditions as described before (47).
Immunofluorescence TC-3 cells (0.1– 0.2 ⫻ 106/well) were plated on poly-l-lysine pretreated (final concentration 0.1 mg/ml) coverslips that were individually placed in each well of a 6-well tissue culture plate (Falcon, Franklin Lakes, NJ). The wild-type mouse pro-GHRH was transiently transfected or cotransfected with either a Golgi marker, enhanced cyan fluorescence protein (pECFP)-Golgi (Clontech Inc., Palo Alto, CA), or a secretory granule marker, enhanced green fluorescence protein (pEGFP)-rPC1/3 (aa 1– 616) (74) according to the Qiagen’s protocol. Also, these two subcellular compartment markers were individually transfected as controls. After transfection, all the cells were fixed in 4% paraformaldehyde in PBS at room temperature for 10 min. Golgi or secretory granule marker transfected cells were stored at 4 C for 2 d in PBS. Cells transfected/cotransfected with pro-GHRH were washed with PBS after fixation and blocked in 5% normal rabbit serum (Jackson ImmunoResearch Laboratories, West Grove, PA) at 4 C overnight. Cells were incubated with the rabbit antimouse GHRH (no. 7802) overnight at 4 C and after PBS washes were incubated with the Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories) for 30 min at room temperature. Finally, after PBS washes, all the anti-GHRH antibody treated and untreated cells were mounted on glass slides and digital images with the help of suitable filters were taken using an Olympus BY 51 fluorescent microscope (Olympus America Inc., Melville, NY) and a CCD camera (DP50, Olympus).
Results and Discussion Processing of mouse pro-GHRH in neuroendocrine cells
The rationale to analyze the roles of furin, PC1/3, PC2, and PC5/6A in pro-GHRH processing are the following: 1) im-
Dey et al. • Processing of Pro-GHRH by PCs
munohistochemical studies have shown that all of these convertases are expressed in the arcuate nucleus of the hypothalamus, the site of GHRH synthesis (7, 8, 17, 64); 2) The amino acid sequence, RXXR2, located just before the N terminus of GHRH in mouse pro-GHRH, is a minimal consensus site for furin cleavage (44, 75, 76) but also has been shown to be cleaved by PC1/3 in some instances (77, 78); 3) PC1/3 and PC2 are known to cleave the sequence RXRXXR2 (78, 79) as is found at the C terminus of GHRH in mouse pro-GHRH; and 4) PC1/3 null mice are growth retarded and have been demonstrated to lack mature GHRH associated with a significant accumulation of unprocessed pro-GHRHlike material in hypothalamic extracts, compared with their wild-type littermates (48). We studied the processing of wild-type mouse pro-GHRH in GH4C1 cells, derived from a rat pituitary tumor. These cells have been reported to contain considerable amounts of both furin and PC5/6A but lack PC1/3 and PC2 (70, 80), which has also been confirmed in our laboratory. Studies following transient transfection/cotransfection and pulsechase labeling (see Materials and Methods for details) show that in absence of any exogenous PCs, wild-type pro-GHRH is processed into a single major peptide, intermediate I, 12– 84aa (Fig. 2, A–C) . It constitutes only 2% of the total levels of pro-GHRH after a 25-min pulse (Fig. 2A, lane 2), 25% in the cells after a 150-min chase (Fig. 2A, lane 3), and 45% of the GHRH immunoreactive peptides in the 150-min chase medium (Fig. 2, B and C, lane 2), indicating significant processing at the N-terminal RXXR site by endogenous furin in the absence of PC1/3 and PC2. We examined the processing of wild-type mouse pro-GHRH using a shorter chase period (30 min) and found very low levels of intermediate I (data not shown), indicating the effectiveness of a longer chase period (150 min) as used in the subsequent experiments. Next, we examined the roles of PC2 or/and PC1/3 coexpression on pro-GHRH processing. In the presence of PC2, we did not find any significant increase in the levels of intermediate I in both cells and medium (Fig. 2, A, lanes 4 and 5, and B and C, lane 3), compared with wild-type alone. However, two other peptides were generated, intermediate II, 1–53aa, and mature GHRH, 12–53aa. These appeared in the 150-min chase medium (Fig. 2C, lane 3) after processing at the C-terminal RXRXXR site of pro-GHRH and intermediate I, respectively. These data suggest that PC2 is unable to process the N-terminal RXXR site but is moderately efficient in cleaving the C terminus (see Fig. 2C, a longer exposure of Fig. 2B). When PC1/3 was cotransfected with wild-type proGHRH, production of intermediate I was considerably increased, especially in the 150-min chase medium, with the ratio of 2:1 between the intermediate I and pro-GHRH (Fig. 2, B and C, lane 4). The results indicate a considerable ability of PC1/3 to cleave the N-terminal site of pro-GHRH. Also, PC1/3 very efficiently cleaved the C-terminal site especially of the predominant intermediate I liberating 64% mature GHRH from the total pool of GHRH immunoreactive peptides after 150-min chase in both cells and medium (Fig. 2, A, lane 7, and B, lane 4). The amounts of mature GHRH produced by PC1/3 relative to PC2 were 4-fold higher (Fig. 2C, lane 4 vs. 3). These findings indicate the significantly greater potency of
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FIG. 2. Processing of wild-type (WT) mouse pro-GHRH in GH4C1 cells. Pro-GHRH expression plasmid (1 g) was transiently transfected/cotransfected (see Materials and Methods for details) without or with mouse PC2 (1 g) or mouse PC1/3 (1 g) expression plasmids. After transfection, cells were pulsed for 25 min with [35S]-Met (150 Ci/ml) and chased for 150 min in presence of 10-fold more cold Met followed by IP of the equal volumes of cell extracts and media with the rabbit anti-GHRH antibody. The immunoprecipitated peptides were resolved in tricine-SDS-PAGE, and after fixation and fluorography, gels were dried and exposed to Hyper films (Amersham Pharmacia Biotech, Piscataway, NJ). A, Pulse (lanes 2, 4, and 6) and chase (lanes 3, 5, and 7) cell extracts (CE). Both B (exposure time, 3 d) and C (exposure time, 10 d) show the corresponding chase media (MED) in lanes 2– 4. Lane 1 for all the panels shows the location of the iodinated authentic GHRH peptide and the positions of the prestained protein molecular weight markers.
PC1/3 in generating mature GHRH. After cotransfection of PC1/3 and PC2 together, we did not find any synergistic effects on the production of mature GHRH (data not shown). It is noteworthy that mouse pro-GHRH (84aa) and intermediate I (12– 84aa) contain five and four Met residues, respec-
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tively, whereas mature GHRH (12–53aa) has only one. Because we used 35S-Met for all of our pulse-chase labeling studies, the net differences in the Met content with reference to band intensities between pro-GHRH and intermediate I and between pro/intermediate I and mature GHRH in cell extracts, and media were taken into consideration for quantitative estimation. Next, we analyzed the importance of the P2 Arg in the N-terminal cleavage site by creating a mutant pro-GHRH, Q10R (see Fig. 1), corresponding to the rat and human sequences (described in detail in Materials and Methods). The presence of a basic residue, Arg/Lys, at the P2 position significantly increased the ability of furin to cleave the site, RXRR, compared with the site lacking the P2 basic residue, RXXR (44, 81), as demonstrated in Fig 3, A and B. We found that 45% of the Q10R pro-GHRH was converted into intermediate I after a 25-min pulse (Fig. 3A, lane 2). After a 30-min chase in cells, 70% of the mutant pro-GHRH was processed into intermediate I (Fig. 3A, lane 3), which ultimately comprised more than 95% of the total levels of GHRH immunoreactive peptides secreted into the 150-min chase medium (Fig. 3B, lane 3). This significant early increase in the Nterminal processing of the Q10R mutant confirms that furin is a potent convertase in producing intermediate I and also implies the existence of a similar processing event for both the rat and human precursors in which both have this P2 Arg
FIG. 3. Processing of N-terminal Q10R mutant with the RXRR cleavage site in GH4C1 cells. The Q10R mutant (1 g) was transiently transfected or cotransfected without or with PC2 (1 g) or PC1/3 (1 g). Cells were pulse labeled for 25 min and chased for two time points, 30 and 150 min. Both the cell extracts (CE) and media (MED) were IP with the rabbit anti-GHRH antibody followed by separation in tricine-SDSPAGE. Lane 1 for all the panels indicates the position of the iodinated authentic GHRH peptide as well as the locations of the prestained protein molecular-weight markers. A, C, and E and B, D, and F were exposed for 3 and 9 d, respectively.
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residue. When we analyzed the individual efficiencies of PC1/3 or PC2 in processing this Q10R mutant, we found, especially in the chase medium, a 30% lower accumulation of intermediate I in the presence of PC1/3, compared with PC2 and, correspondingly, 6-fold higher levels of the mature GHRH relative to PC2 (Fig. 3F, lane 3 vs. 3D, lane 3). The total amount of mature GHRH liberated by PC1/3 in both cells and medium after 150 min of chase was 67% of the total levels of GHRH immunoreactive peptides (Fig. 3, E, lane 4, and F, lane 3). Mature GHRH, liberated due to coexpression of PC1/3 and the Q10R mutant, comprised 45% of the total secreted anti-GHRH reactive peptides at 150 min relative to 36% from the wild-type prohormone (Fig. 3 vs. Fig. 2). Increased accumulation of mature GHRH by PC1/3 as opposed to PC2 was also observed in the cells after a 150-min chase (Fig. 3E, lane 4 vs. Fig. 3C, lane 4). These data confirm the greater ability of PC1/3 to cleave the C-terminal RXRXXR site to generate mature GHRH. To further examine the role of furin in processing the N-terminal RXXR site of wild-type pro-GHRH, we employed a furin null Chinese hamster ovary cell line, RPE.40 (82). As shown in Fig. 4, after transfection of wild-type pro-GHRH, no processing was observed even after a long chase (5 h) in both cell extracts and medium (Fig. 4, A, lanes 2–5, and B, lanes 2– 4). When furin was cotransfected, 50% of pro-GHRH was processed into intermediate I during the chase period in
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FIG. 4. Processing of WT mouse proGHRH in RPE.40 cells. Cells were transiently transfected/cotransfected with pro-GHRH plasmid DNA (1 g) without or with human furin expression plasmid (1 g). After pulse-chase labeling, cell extracts (both pulse and chase) and chase media of equal volumes were IP with the rabbit anti-GHRH antibody. The immunoprecipitated peptides after separation in tricine-SDS-PAGE were fixed and fluorographed and finally exposed to Hyper films. Both A and C show the cell extracts (CE) of pulse and chase without or with furin, respectively. B and D show the corresponding chase media (MED). Lane 1 shows the position of the iodinated authentic GHRH peptide and the locations of the prestained molecular-weight markers.
cells beginning at early time points (Fig. 4C, lanes 3 and 4) after a 25-min pulse (Fig. 4C, lane 2). In the chase medium, intermediate I constituted more than 95% of total secreted peptides (Fig. 4D, lanes 3 and 4). However, we found only low levels of mature GHRH in the presence of exogenous furin (Fig. 4, C and D), indicating the very limited ability of this convertase to cleave the C terminus of both pro-GHRH and intermediate I in RPE.40 cells. In similar experiments, using another furin defective cell line, LoVo, derived from a human colon carcinoma (83), we found significant generation of intermediate I from pro-GHRH by exogenous furin (data not shown). Employing a furin inhibitor, decanoylArg-Val-Lys-Arg-chloromethylketone (Bachem, Torrance, CA) (44), we observed a significant inhibition of the Nterminal processing of wild-type pro-GHRH in GH4C1 cells in absence of any exogenous PCs (data not shown). All these results strongly suggest that furin can be a key convertase in cleaving the N-terminal RXXR/RXRR site of pro-GHRH. Next, we examined the role of PC5/6A on pro-GHRH processing with the help of a mouse neuroblastoma cell line, Neuro-2A, having undetectable or very low levels of PC5/ 6A, PC2, and PC1/3 but considerable amounts of furin (80). We used the Q10R mutant because of its much greater susceptibility to furin-mediated cleavage at the N terminus in generating significantly higher levels of intermediate I (Fig. 3) relative to the wild-type pro-GHRH (Fig. 2). We showed that the mutant pro-GHRH, when transfected alone, was significantly cleaved at its N terminus after a 25-min pulse producing high levels of intermediate I, and it constituted 40% of the total amounts of proform (Fig. 5A, lane 2). After the 150-min chase, the levels of intermediate I in cells were considerably augmented, reaching a ratio of 1:1 (Fig. 5A, lane 3). In the 150-min chase medium, the predominant peptide was intermediate I (Fig. 5A, lane 4), suggesting that the N-terminal RXRR site of the Q10R mutant was efficiently cleaved by endogenous furin in Neuro-2A cells. After cotransfection with PC5/6A, we found a modest increase in
N-terminal processing after the 25-min pulse amounting to 50% conversion of pro-GHRH into intermediate I (Fig. 5A, lane 5). This N-terminal cleavage was further enhanced during the chase, especially within the cells in which the ratio of intermediate I to pro-GHRH rose to 2:1 (Fig. 5A, lane 6). We did not find any detectable levels of mature GHRH under these conditions (Fig. 5A, lanes 5–7). The results indicate a modest but significant role of PC5/6A in cleaving at the N-terminal RXRR site but no evidence for cleavage at the C-terminal site of either intermediate I or pro-GHRH by this convertase. When PC5/6A was coexpressed with wild-type pro-GHRH in GH4C1 cells, the levels of intermediate I were clearly augmented in both cells and medium, especially after 150 min chase, compared with pro-GHRH alone (Fig. 5B, lanes 6 and 7 vs. 3 and 4). After cotransfection of a furin inhibitor, ␣1-PDX (44), processing of pro-GHRH into intermediate I was significantly blocked (Fig. 5C, lanes 2– 4). To examine the ability of PC5/6A in counteracting the furininhibitory action of ␣1-PDX, we coexpressed PC5/6A with both ␣1-PDX and wild-type pro-GHRH and showed that PC5/6A was able to generate significant amounts of intermediate I, confirming its ability to process the N-terminal RXXR/RXRR cleavage site (Fig. 5C, lanes 6 and 7). To understand the importance of each of the Arg residues in both the N- and C-terminal cleavage sites, we individually mutated the arginines at P1 and P4 of the N terminus and P1, P4, and P6 of the C terminus to Ala (see Fig. 1 and Materials and Methods). We demonstrated in cotransfection assays in GH4C1 cells that for both the P1 mutants, R11A and R54A, processing was significantly blocked at the N and C terminus, respectively, leading to total loss of mature GHRH synthesis in the presence of PC1/3 (see Figs. 6B and 7, A and B, lanes 8 –10). We found considerable enhancement of intermediate II levels when R11A was coexpressed with PC1/3 (Fig. 6B, lanes 5–7 vs. lanes 2– 4), indicating no apparent impairment of the C-terminal processing of this mutant by PC1/3. Generation of very low levels of intermediate II from
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FIG. 6. Processing of N-terminal mutants of mouse pro-GHRH in GH4C1 cells. A, Mutant R8A expression plasmid (1 g) was transfected without or with PC1/3 (1 g), whereas in B, mutant R11A (1 g) was transfected in absence or presence of PC1/3 (1 g). After metabolic pulse-chase labeling and immunoprecipitation of both the cell extracts (CE) and media (M), peptides were resolved in tricine gels followed by fixation and fluorography. Lane 1 represents the authentic GHRH peptide location and the positions of the prestained markers.
FIG. 5. Processing of mouse pro-GHRH in Neuro-2A and GH4C1 cells. A, Neuro-2Acells were transiently transfected/cotransfected with the N-terminal mutant pro-GHRH, Q10R, expression plasmid (1 g) without or with mouse PC5/6A plasmid DNA (1 g). B, GH4C1 cells were transfected/cotransfected with WT pro-GHRH (1 g) without or with PC5/6〈 (1 g). C, WT pro-GHRH was cotransfected either with ␣1-PDX (1 g) or PC5/6A and ␣1-PDX together (1 ⫹ 1 g) in GH4C1 cells. After metabolic pulse-chase labeling, cell extracts (CE) and media (M) were IP with the rabbit ant-GHRH antibody and the immunoprecipitated peptides were resolved in tricine gels. Lane 1 shows the locations of the authentic GHRH peptide as well as the prestained protein markers.
R11A in the absence of PC1/3 confirms that the C-terminal site is a poor substrate for furin. No other intermediate cleavage products were detected. When the N-terminal P4 mutant, R8A, was cotransfected with PC1/3, both intermediate I and mature GHRH were generated and appeared in the 150-min chase medium after processing at the N-terminal P1 Arg and the C-terminal site, respectively, along with a very low level
of unprocessed pro-GHRH (Fig. 6A, lane 7). The diminished levels of both intermediate I and mature GHRH indicate the requirement of a P4 Arg for efficient processing of the N terminus. Processing at a single Arg residue by PC1/3, as observed for the R8A mutant, has been shown before (47, 60, 78). Similar to R11A, very low levels of intermediate II were generated from R8A in absence of exogenous PC1/3 (6A, lanes 2– 4), confirming again the very limited ability of endogenous furin to process the C-terminal site of both the N-terminal mutants in GH4C1 cells. We did not find any detectable cleavage of either of the N-terminal mutants, R11A or R8A, in presence of exogenous furin (data not shown), confirming a requirement of the minimal consensus RXXR sequence for furin action (44, 75). In GH4C1 cells, the C-terminal P1 mutant, R54A, when transfected alone, was considerably processed at its N terminus to generate intermediate I (Fig. 7A, lanes 9 and 10). When coexpressed with PC1/3, increased amounts of intermediate I were generated from R54A and secreted into the chase medium along with a very low level of unprocessed proform (Fig. 7B, lanes 9 and 10). The results suggest that the C-terminal P1 Arg to Ala mutation did not significantly affect the N-terminal processing. Similar to R54A, generation of intermediate I was not significantly affected for either of the other two C-terminal mutants, R51A or R49A, when expressed alone (Fig. 7A), indicating again a lack of influence of C-terminal site structure on processing at the N-terminal RXXR site. We showed in cotransfection assays using either of the mutants, R51A or R49A, that either of the Arg residues, P4 and P6, was required for complete processing of the C
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terminus to generate maximum levels of mature GHRH in the presence of PC1/3 (Fig. 7B, lanes 3 and 4 and 6 and 7). Subcellular localization of GHRH immunoreactive peptides
We used TC-3 cells, derived from mouse pancreatic islets, to study the localization of anti-GHRH antibody reactive peptides after transient transfection/cotransfection of wildtype pro-GHRH without or with Golgi and secretory granule markers, pECFP-Golgi and pEGFP-PC1/3, respectively (see details in Materials and Methods). Our immunocytochemical analyses showed the distribution of GHRH or its precursors in the Golgi as indicated by Cy3 fluorescence alone (Fig. 8A, panel a) and combined fluorescence of ECFP ⫹ Cy3 (Fig. 8A, panel c). Both of these localization signals were comparable
FIG. 7. Processing of C-terminal mutants of mouse pro-GHRH in GH4C1 cells. Cells were either transfected with 1 g of each of the C-terminal mutants, R49A, R51A, and R54A (A) or cotransfected also with PC1/3 (1 g) (B). Cells were pulsed for 25 min and chased for 150 min followed by IP of both the cell extracts (CE) and media (M) with the rabbit anti-GHRH antibody. Lane 1 shows the locations of the iodinated authentic GHRH peptide and the prestained markers.
FIG. 8. Immunocytochemical localization of WT pro-GHRH in Golgi and secretory granules in TC-3 cells. Cells were transiently transfected with either 500 ng of WT pro-GHRH (A and B, panel a) or 500 ng of pECFP-Golgi (A, panel b) or 1 g of pEGFP-PC1/3 (B, panel b). WT pro-GHRH was cotransfected either with pECFP-Golgi (A, panel c) or with pEGFP-PC1/3 (B, panel c). The white arrows in panels a to c of A and B indicate the Golgi and secretory granules, respectively. (Magnifications: A, ⫻400; B, ⫻800.)
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with pECFP-Golgi-generated fluorescence (Fig. 8A, panel b), showing the likely presence of GHRH precursor peptides in the Golgi after synthesis in the endoplasmic reticulum. We also showed the localization of GHRH immunoreactive peptides in the secretory granules as indicated by Cy3 alone (Fig. 8B, panel a) and the combined Cy3 and EGFP fluorescence (Fig. 8B, panel c), both of which were comparable with the staining generated by EGFP alone (Fig. 8B, panel b). A model for pro-GHRH processing
We propose (see Fig. 9) that pro-GHRH, during its transit through Golgi 3 TGN 3 ISGs in the regulated secretory pathway is first cleaved at its N-terminal site by the combined actions of furin, PC1/3, and PC5/6A (furin ⬎⬎
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FIG. 9. A model illustrating mouse pro-GHRH processing in neuroendocrine cells. This processing scheme for mouse pro-GHRH was derived from the analyses of data of transient transfection/cotransfection of wild-type and several point mutants of mouse pro-GHRH, pulse-chase labeling and immunocytochemistry. The two major cleavage sites of mouse pro-GHRH, the N-terminal RMQR and C-terminal RARLSR, are shown on the left and right side, respectively. The blue arrow represents those prohormone convertases involved in the Nterminal processing, whereas the green arrow indicates the convertases responsible for the C-terminal cleavage. The bold and lighter black arrows indicate major vs. minor pathways for mature GHRH synthesis.
PC1/3 ⬎ PC5/6A) to generate significant amounts of intermediate I, 12– 84aa indicated by the blue and bold black arrows). We designated intermediate I as the major intermediate due to its high abundance. Processing then occurs at the C-terminal cleavage site catalyzed most efficiently by PC1/3 and weakly by PC2 to produce mature GHRH, 12–53aa (indicated by the green and bold black arrows). Our model also shows that PC1/3 acting alone can mediate processing of the N and C termini of pro-GHRH to generate substantial levels of mature GHRH (marked by a lighter black arrow) and small amounts of intermediate II, 1–53aa (the minor intermediate). Also, the model depicts that low levels of intermediate II can be generated by PC2 due to processing at the C-terminal site of pro-GHRH (indicated by a lighter black arrow). In studies with ␣TC1– 6 cells having high levels of endogenous PC2 and undetectable amounts of PC1/3 (60), we found low levels of both the intermediate II and mature GHRH generated from pro-GHRH (data not shown). Using a C-terminally truncated cDNA generating intermediate II, we found in TC-3 cells with high levels of both PC2 and PC1/3 (80) that it
Dey et al. • Processing of Pro-GHRH by PCs
was efficiently converted to mature GHRH (data not shown). The results altogether have indicated that PC1/3 is the most efficient convertase to liberate mature GHRH via two pathways: major, processing of the C-terminal site of intermediate I, which is generated from pro-GHRH via N-terminal cleavage mediated most efficiently by furin and partly by PC5/6A (furin ⬎⬎ PC5/6A); and minor, processing by PC1/3 of both sites in pro-GHRH. The results also indicate that PC1/3 may have a greater preference for intermediate I as a substrate than pro-GHRH probably because of a more favorable conformation of intermediate I after the removal of the N-terminal 11aa segment from pro-GHRH by the earlier action of furin. Our data suggest not only an overlapping function of these three convertases in targeting the same N-terminal cleavage site but also a mechanism to ensure the efficient processing of pro-GHRH at its N terminus during its transit through Golgi 3 TGN 3 ISGs in the regulated secretory pathway after its synthesis in endoplasmic reticulum. Recent studies have shown that PC5/6A is able to cleave proneurotensin/neuromedin at a dibasic Lys-Arg site and pro-cholecystokinin at a monobasic Arg/Lys site in the early compartments of the regulated secretory pathway (63, 65, 84). PC5/6 has also been shown to cleave the RXXR site of prorenin in neuroendocrine cells (24). Our data on proGHRH processing indicate for the first time that PC5/6A is able to process an RXXR/RXRR site of a hypothalamic prohormone in the regulated secretory pathway. To determine that pro-GHRH processing is a stepwise event rather than simultaneous, we showed in GH4C1 cells that high levels of intermediate I were generated after 25-min pulse labeling of the N-terminal mutant, Q10R (Fig. 3A, lane 2). The levels of intermediate I were significantly enhanced because the mutant was chased for a period of 30 min without any exogenous PCs (Fig. 3A, lane 3, and 3B, lane 2). These results corroborate with the previous findings on furin action in the early compartments (44, 81). On the other hand, we found low levels of mature GHRH after a 30-min chase in the presence of PC1/3 (Fig. 3E). As the chase time was extended, 6-fold more mature GHRH were generated from the Q10R mutant by PC1/3, compared with PC2 (PC1/3 ⬎⬎ PC2) (see Fig. 3, D, lane 3, and F, lane 3), confirming actions of both PC1/3 and PC2 later than furin, probably predominantly in secretory granules within the regulated secretory pathway (18, 40, 78). Similarly, for the wild-type pro-GHRH, we showed the late actions of both PC1/3 and PC2 (PC1/3 ⬎⬎ PC2) in generating mature GHRH (Fig. 2, A, lanes 5 and 7, and B and C, lanes 3 and 4). In AtT20 cells (endogenous PC1/3 ⬎⬎⬎ PC2) (60, 80), as expected, we found significant generation of mature GHRH from pro-GHRH. It is noteworthy that furin level was reported low in AtT20 cells (80) as we also found in our laboratory (data not shown). Also, our immunocytochemical studies showing localization of GHRH immunoreactive peptides in Golgi and secretory granules in TC-3 cells (Fig. 8, A and B) corroborate with the subcellular sites for the actions of these convertases. All these results, using several neuroendocrine cells, along with those reported by others in a recent preliminary report (85), confirm the probable biological relevance of our studies.
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On the basis of our current findings, we believe that the high-molecular-weight forms, present in the PC1/3 knockout mouse hypothalamic extracts, may represent both unprocessed pro-GHRH, 84aa, and intermediate I, 12– 84aa, possibly generated by furin. Western blot analysis of hypothalamic extracts to identify GHRH immunoreactive peptides has so far been unsuccessful (our unpublished data). To determine the exact role of furin in hypothalamic pro-GHRH processing, targeted disruption of furin expression in hypothalamus will be required because both furin and PC1/3 are present in the arcuate nucleus of the hypothalamus (17), the site of GHRH synthesis (7, 8). The underlying causes of the major phenotypic differences between mice (48) and a human subject (86) lacking active PC1/3 are currently not well understood. Possible reasons include species differences in convertase expression or a greater vulnerability of the N-terminal RXRR site of human pro-GHRH to furin-mediated cleavage due to the presence of the P2 Arg in the human precursor as shown here. On the basis of our current results, we can propose two hypotheses to account for the normal somatic growth of the human subject lacking active PC1/3: 1) PC2 or some other PCs such as PC5/6A may be able to generate sufficient mature GHRH via C-terminal processing of the elevated levels of intermediate I to elicit normal growth-regulatory responses, or 2) because a free N terminus of GHRH might be required for maximum biological potency (87, 88), the partially active intermediate I alone at high levels might be able to compensate for the absence of mature GHRH. Acknowledgments We thank Paul Gardner for making the oligonucleotide primers and Margaret Milewski and Rohan Shah for their technical assistance in cell culture. We also thank Rosie Ricks for expert secretarial assistance. Received October 31, 2003. Accepted December 8, 2003. Address all correspondence and requests for reprints to: Donald F. Steiner, Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637. E-mail: dfsteine@midway. uchicago.edu. This work was supported by NIH Grant DK13914 and the Howard Hughes Medical Institute.
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Twenty-Third Annual University of Kentucky Symposium in Reproductive Sciences May 20 –21, 2004 This annual symposium of the Reproductive Sciences Forum at the University of Kentucky brings faculty, clinicians, and trainees in the field of reproductive sciences together to exchange information and ideas with each other and with recognized leaders of the reproductive sciences research community. The program consists of plenary lectures, a poster session, meet-the-professor luncheon and dinner. For registration information visit our website at: http://www.mc.uky.edu/obg/forum/symposium/home.htm Or contact: Tae Ji, Ph.D. Symposium Director Department of Chemistry University of Kentucky Lexington, KY 40506 Tel: (859) 257-3163
[email protected] Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.
