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Endocrinology Vol. 140, No. 12 5817-5827
Copyright © 1999 by The Endocrine Society

ARTICLES

Posttranslational Processing of Progrowth Hormone-Releasing Hormone1

Eduardo A. Nillni, Rosemary Steinmetz and Ora Hirsch Pescovitz

Division of Endocrinology, Department of Medicine, Brown University School of Medicine, Rhode Island Hospital (E.A.N.), Providence, Rhode Island 02903; and the Section of Pediatric Endocrinology and Diabetology, The Herman B. Wells Center for Pediatric Research, Departments of Pediatrics and Physiology, James Whitcomb Riley Hospital for Children, Indiana University (R.S., O.H.P.), Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Eduardo A. Nillni, Division of Endocrinology, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The prepro-GH-releasing hormone (prepro-GHRH; 12.3 kDa) precursor, like other neuropeptide precursors, undergoes proteolytic cleavage to give rise to mature GHRH, which is the primary stimulatory regulator of pituitary GH secretion. In this study we present the first model of in vitro pro-GHRH processing. Using pulse-chase analysis, we demonstrate that at least five peptide forms in addition to GHRH are produced. The pro-GHRH (after removal of its signal peptide, 10.5 kDa) is first processed to an 8.8-kDa intermediate form that is cleaved to yield two products: the 5.2-kDa GHRH and GHRH-related peptide (GHRH-RP; 3.6 kDa). GHRH-RP is a recently described peptide derived from proteolytic processing of pro-GHRH that activates stem cell factor, a factor known to be essential for hemopoiesis, spermatogenesis, and melanocyte function. Further cleavage results in a 3.5-kDa GHRH and a 2.2-kDa product of GHRH-RP.

Like GHRH, there is GHRH-RP immunostaining in hypothalamic neurons in the median eminence as detected by immunohistochemistry and immunoelectron microscopy. Based on deduced amino acid sequences of the pro-GHRH processing products, several peptides were synthesized and tested for their ability to stimulate the cAMP second messenger system. GHRH, GHRH-RP, and one of these peptides [prepro-GHRH-(75–92)-NH2] all significantly stimulated the PKA pathway. This work delineates a new model of pro-GHRH processing and demonstrates that novel peptides derived from this processing may have biological action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY NEUROPEPTIDES and neurotransmitters are first synthesized as large proprotein precursors. After their synthesis in the rough endoplasmic reticulum (RER), these prohormones or proneurotransmitters are posttranslationally modified to give rise to mature peptides that have unique biological actions. Posttranslational processing of proteins involves diverse mechanisms, including phosphorylation, glycosylation, and endoproteolytic processing. Limited endoproteolytic cleavage occurs at paired basic residues, either Lys-Arg or Arg-Arg, with cleavage at monobasic sites occurring less frequently (1, 2, 3). For many prohormones, serial processing occurs as they are targeted to the regulated secretory pathway. In the neuroendocrine system, it is believed that fully processed bioactive peptides are stored in secretory granules that are released only after ligand-specific stimulation of a membrane-bound receptor (4). There is a significant body of work describing the enzymes involved in the posttranslational processing of many neuropeptides, including POMC (5, 6), pro-TRH (1, 7, 8), proinsulin (9), procholecystokinin (10), and proenkephalin (11). However, to date, the products generated and the mechanisms involved during posttranslational processing of prepro-GH-releasing hormone (pro-GHRH) have not been studied.

GHRH is the primary hypothalamic factor responsible for the synthesis and secretion of GH from anterior pituitary somatotrophs (12, 13, 14). GHRH also has mitogenic and cell-differentiating effects in somatotrophs in vitro (15). In rat hypothalamus, the GHRH neurons involved in somatotroph regulation originate from the arcuate nucleus in the ventrolateral hypothalamus and project to the external layer of the median eminence, where they terminate in close proximity to the hypophysial portal circulation (16, 17, 18, 19, 20). A recent study using autoradiographic in situ hybridization reveals that the GHRH messenger RNA is also detected throughout the ventromedial hypothalamic nucleus and the ventromedial aspect of the arcuate nucleus (21).

Despite a large body of work defining the biological role of GHRH in the regulation of GH secretion, little is known about the biosynthesis and posttranslational processing of pro-GHRH. Pro-GHRH shares considerable homology with the precursors for both vasoactive intestinal peptide and pituitary adenylate-activating peptide, which during processing yield at least two physiologically significant peptides. We hypothesized that proteolytic processing of pro-GHRH might result in the production of more than one bioactive peptide. To begin to address this possibility, we postulated that a peptide product was produced from the carboxyl-terminal region of the GHRH precursor, corresponding to prepro-GHRH-(75–104). We therefore had this peptide, GHRH-related peptide (GHRH-RP), synthesized. In preliminary in vitro studies in Sertoli cells, we demonstrated that GHRH-RP is a potent stimulator of stem cell factor (SCF), a germ cell factor that is critical for normal spermatogenesis (22). In addition, overexpression of GHRH-RP in a transgenic mouse model increases SCF expression in numerous tissues (23) and also increases turnover of bone marrow and spleen hemopoietic precursors (24). The current work is designed to characterize the biosynthesis and processing of pro-GHRH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). On day 17 of gestation, after pentobarbital-anesthesia (60 mg/kg) was administered, the abdominal cavity was opened, and the fetuses were removed. Each fetus was decapitated, and the diencephalon was removed (25). Experimental protocols and euthanasia procedures were approved by the Rhode Island Hospital/Brown University and the Indiana University institutional animal care and use committees.

Hypothalamic cultures
Cultures were undertaken as previously described (25). In brief, diencephalic tissue was dissociated to single cells by neutral protease digestion (1 U/tissue; Sigma, St. Louis, MO). The cells were cultured for up to 14 days in DMEM L-15 (26) containing 10% FCS (Life Technologies, Inc., Gaithersburg, MD) and supplemented with various additives. Before plating, all wells were coated with poly-D-lysine (20 µg/ml; Sigma). To induce differentiation of neuronal cells and pro-GHRH biosynthesis, the cells were cultured with 50 µM bromodeoxyuridine during the first 4 days as previously described (25). For immunocytochemistry (ICC) and immunoelectron microscopy (IEM), the cells were plated on four-chamber glass Lab-Tek (Nunc, Inc., Naperville, IL) slides (106 cells/ml). For radiolabeling experiments, the cells were incubated in 25-cm2 flasks (5–6 x 106 cells/flask).

Radiolabeling and peptide extraction
On day 12 in culture, each flask of hypothalamic neurons containing 5 x 106 cells/flask was pulsed with 0.7 mCi [3,4,5-3H]leucine (156 Ci/mmol) in leucine-free DMEM or 750 µCi L-[35S]methionine (1175 Ci/mmol) containing 3% FCS for 30 min. Cells were treated with control medium or with 10-7 M phorbol 12-myristate ester (PMA) for 45 min. After the incubations, the medium was removed, and the cells were washed three times with Hanks’ buffer containing 0.1 mg/ml cold leucine. After the last wash, the cells were rapidly cooled on ice, and 2 ml 2 N acetic acid containing 2 mM EDTA, 2 mM EGTA, and enzyme inhibitors (phenylmethylsulfonylfluoride, aprotinin, bacitracin, bestatin, and pepstatin, each at 0.1%) were added. The cells were scraped and heated to 95 C for 10 min before sonication. One hundred microliters of sample were removed for protein assay. The remainder of the cell extract was centrifuged at 15,000 rpm for 30 min. The supernatant was then lyophilized and held at -20 C until analyzed by PAGE.

Synthetic peptides
GHRH-RP was synthesized in the Biochemistry Biotechnology Facility at Indiana University from the deduced amino acid sequence of the prepro-GHRH peptide (corresponding to amino acids 75–104 of the precursor) and purified by HPLC. The following peptides were synthesized by the American Peptide Co. (Sunnyvale, CA): 1) HLDRVWAED, designated prepro-GHRH-(75–83); and 2) HLDRVWAEDKQMALESIL-NH2, designated prepro-GHRH-(75–92)-NH2, with an amide group on the C-terminal leucine.

Antibodies
GHRH-RP was conjugated to BSA, and antisera were produced in rabbits (Hazelton Laboratories, Vienna, VA) (22). The resultant GHRH-RP antisera were assayed and characterized in an enzyme-linked immunosorbent assay system and were shown to be of high titer and specificity for GHRH-RP. No cross-reactivity with rat GHRH, vasoactive intestinal peptide, pituitary adenylate-activating peptide, secretin, PHI, or glucagon was seen at concentrations up to 1 mg. The GHRH antisera used in these studies have previously been described and were demonstrated to be effective in immunoassays (27) and in their ability to neutralize pituitary GH secretion (28).

Immunoprecipitation and SDS-PAGE
The immunoprecipitation protocol for pro-GHRH was carried out as previously described (7). The immunoprecipitates were then resuspended in sample buffer [0.0625 M Tris (pH 6.8), 1% SDS, 15% glycerol, and 15 mM dithiothreitol] and boiled for 4 min before SDS-PAGE. Samples were analyzed by loading them onto a 1.5-mm discontinuous polyacrylamide gel tricine-SDS system. Gels were run in the Protean 16-cm cell system (Bio-Rad Laboratories, Inc., Richmond, CA). After electrophoresis, gels were cut into 2-mm slices in a gel slicer (Hoeffer Scientific, San Francisco, CA) and prepared for counting. Immunoprecipitated peptides were extracted from the gel slices by incubation in 0.5 ml 2 N acetic acid for 24 h at 4 C. Scintillation fluid (Bio Safe II, RPI, Mount Prospect, IL) was added, and the samples were counted in a scintillation counter. Prestained molecular mass markers (Bio-Rad Laboratories, Inc.: 48.3, 33.4, 28.3, and 19.4 kDa; Diversified Biotech: 29, 20.4, 14.4, 6.5, and 2.8 kDa) were used [Diversified Biotech (Newton, MA) and Bio-Rad Laboratories, Inc. (Richmond, CA)].

Immunohistochemistry (IHC), immunocytochemistry (ICC), and immunoelectronmicroscopy (IEM)
The hypothalamic distribution of GHRH-RP was determined by IHC. Adult rat brains were perfused at sacrifice with 4% paraformaldehyde and then sectioned (35 µm). The tissue was incubated with rabbit anti-GHRH-RP serum (1:2000), followed by second antibody treatment with biotinylated goat antirabbit IgG (1:200). Avidin DH-biotin-peroxidase complex (Vector Laboratories, Inc., Burlingame, CA) was used as substrate, and the slides were developed in a 3,3'-diaminobenzadine (DAB)-hydrogen peroxide solution. Controls included substitution of preimmune serum for primary antisera and antisera preadsorbed with excess antigen (100 µM GHRH-RP).

For ICC in hypothalamic neurons, the cells (3 x 105 cells/well) were cultured in four-chamber Lab-Tek slides (Nalge, Nunc, Inc.) for 12 days and fixed with 4% paraformaldehyde in PBS. After three washes with PIPES/sucrose buffer (0.1 M PIPES, pH 6.8, and 0.12 M sucrose), the cells were incubated in 0.2% Triton X-100 in the same buffer. After blocking with 5% albumin, the cells were washed with PBS containing 0.1 M NH4Cl and 1% normal goat serum. Immunoreaction with both primary antisera (1:1500) was performed at 4 C for 24 h. Goat antirabbit Ig (1:2000) conjugated with fluorescein isothiocyanate was used as the fluorescence marker, with an incubation time of 4 h at 4 C for the secondary antibody. Control experiments included the incubation of cells without primary antibody or preimmune serum and the blocking of the primary antibody with the synthetic GHRH and GHRH-RP peptides. For IEM, cells were fixed as described above, and a preembedding peroxidase-DAB procedure was performed.

Sertoli cell cultures and cAMP activation studies
Sertoli cells were isolated from 20- to 22-day-old male Wistar rats as previously described (29). Essentially, whole testes were removed and decapsulated, and seminiferous tubules were isolated by collagenase digestion (1 mg/ml in Ham’s F-12 medium at 37 C for 10 min). Seminiferous tubules were carefully washed with HBSS, then transferred to a sterile dish, minced, treated with trypsin (0.25% in HBSS for 10 min at room temperature), washed, and redigested with collagenase for 30–40 min. Cells were centrifuged (400 rpm for 2 min), washed twice with HBSS, and resuspended in serum-free Ham’s F-12 medium supplemented with antibiotics and fungizone. Cells, a mixture of Sertoli, peritubular, and germ cells, were plated at 4 x 105cells/well in six-well tissue culture plates and incubated for 4 days at 32 C, with fresh medium added on day 3. During this time, only the Sertoli cells survive; the peritubular and germ cells die due to the serum-free conditions.

For determination of cAMP activation, the PathDetect CREB trans-reporting system (Stratagene, La Jolla, CA) was used. This system measures the magnitude of cAMP-specific activation by either intracellular or extracellular stimuli. For this, cells were transfected for 5–6 h, using Lipofectamine (Life Technologies, Inc.; 4 µg/well), with the DNAs supplied in the PathDetect kit and following the manufacturer’s protocol. After transfection, fresh medium (Ham’s F-12 containing 1% FBS) was added, and the cells were incubated for an additional 18 h, at which time the following treatments were added: GHRH, GHRH-RP, prepro-GHRH-(75–83), and prepro-GHRH-(75–92)-NH2 (100 nM each). Forskolin (10 µM) was used as a positive control, and PMA (50 nM) served as a negative control. Cells transfected with pFR-luc (reporter plasmid alone) and pFC-PKA (internal positive control supplied by the manufacturer) served as transfection controls. After 7 h of treatment, cells were lysed, luciferase activity was measured using the Promega Corp. Luciferase Assay System (Madison, WI), and results were normalized to total protein (BCA Protein Assay Kit, Pierce Chemical Co., Rockford, IL). Each experiment contained three transfections per treatment group and was repeated three or four times. ANOVA with Duncan’s post-hoc testing was performed using Dataxiom Statistical Software, Inc. (DataMost Corp, Los Angeles, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of immunoreactive peptides using anti-GHRH-RP in rat hypothalamus
Intense and specific GHRH-RP staining was localized to the nerve terminals and fibers in the median eminence (Fig. 1Go). No immunoactivity could be demonstrated in other brain regions, and no signal was detected in brains stained with preimmune rabbit serum. Further confirmation of specificity of the signal was provided by the complete obliteration of staining in samples treated with GHRH-RP antisera that had been preincubated with an excess of synthetic GHRH-RP. Moreover, preincubation of the GHRH-RP antisera with an excess of synthetic GHRH did not diminish the immunohistochemical signals. The distribution pattern throughout the external lamina of the median eminence observed in this study using anti-GHRH-RP was similar to that observed previously when using anti-GHRH (30). These results suggest that products containing the carboxyl-terminal region of pro-GHRH, in addition to mature GHRH, reach the median eminence where they are positioned for release into the portal circulation.



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  		Figure 1. Immunohistochemical localization of GHRH-RP in rat hypothalamus. Coronal sections of rat hypothalamus showing the third ventricle and median eminence. Sections were incubated with preimmune antiserum (A), GHRH-RP-specific antiserum (B), or preadsorbed GHRH-RP antiserum (C). Original magnification, x100 for all panels. Hypothalamic sections were counterstained with methyl green.

 
Anatomical localization of pro-GHRH-derived peptides in hypothalamic neurons in tissue culture
To investigate the subcellular distribution of pro-GHRH and its products, hypothalamic neurons were cultured for 12–14 days in vitro. After 14 days, the hypothalamic neurons demonstrated an increased outgrowth of neurites similar in morphology to peptidergic neurons in vivo. Most of the neurons were bipolar, with long axons containing varicosities, boutons, and growth cones. Many of the growth cones were in contact with neurites of other neurons. In addition, dendrite-like structures were observed. The anatomical distribution of pro-GHRH and its derived peptides was determined in these cultures by ICC using anti-GHRH and anti-GHRH-RP sera. No more than 5% of the neurons were immunoactive with either antiserum, in agreement with known data indicating a paucity of GHRH neurons in rodent hypothalami (<3000 GHRH neurons in the entire rat arcuate nucleus) (30). Using protein G-purified anti-GHRH and anti-GHRH-RP, we were able to identify positive immunostaining with both antisera (Fig. 2Go), making these cultured neurons a suitable endogenous system for processing studies. Positive staining was observed in cell bodies and neurites with both antibodies. To ascertain whether immunoreactivity to anti-GHRH is associated with organelles of the regulated secretory pathway, IEM analysis of hypothalamic neurons was performed (Fig. 3Go). Positive staining was observed in the RER, Golgi complex and secretory granules (SGs), indicating that pro-GHRH and its derived peptides are present in cellular compartments of the regulated secretory pathway.



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  		Figure 2. Immunocytochemical localization of pro-GHRH-derived peptides in primary cultures of hypothalamic neurons. Neuronal cells cultured for up to 14 days in four-chamber Lab-Tek slides were fixed with 4% paraformaldehyde followed by immunoreaction with anti-GHRH or anti-GHRH-RP. Fluorescein isothiocyanate conjugated to goat antirabbit globulin was used as a probe. Low (A, x100) and high (B, x400) magnifications are shown. A is a low magnification of neurons immunostained with anti-GHRH indicating staining in the cell bodies (arrowhead) and neurites; note a negative cell (arrow). B is a high magnification of neurons stained with anti-GHRH demonstrating immunoactivity in cell bodies (arrows) and neurites (arrowheads). C is a high magnification of neurons stained with anti-GHRH-RP, with positive staining in cell bodies (arrows) and neurites (arrowheads). A representative nonstained dendrite from an immunopositive cell is shown in Fig. 3BGo. These data support the concept that after biosynthesis of proneuropeptides in the RER, they are targeted to the axonal processes for later release.

 


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  		Figure 3. Subcellular location of pro-GHRH and its derived peptides in primary cultures of hypothalamic neurons. The cells were fixed with 4% paraformaldehyde, and positive staining was visualized using a peroxidase-DAB staining reaction. A, Low magnification of hypothalamic neurons positively stained with anti-GHRH. B, Higher magnification of positively stained SGs (arrowheads). C, Higher magnification of an immunopositive neuron with staining in the cytoplasm near the Golgi complex and in the RER (arrowheads). SGs near the nucleus and in the axonal processes are also positively stained (B and D). D, Positively stained neurite with anti-GHRH (arrows) next to a negative neurite from a different neuron (arrowheads).

 
Pulse-chase studies
To determine how pro-GHRH is processed to its intermediate forms and mature peptides, we performed a set of pulse-chase experiments in primary cultures of hypothalamic neurons, using [3H]leucine and [35S]methionine as isotopes. Leucine was chosen because of its occurrence (nine times) throughout the pro-GHRH molecule, and methionine because it is found at amino acid position 57 in the GHRH sequence and at positions 86 and 98 in GHRH-RP. Figures 4–7GoGoGoGo depict representative pulse-chase profiles for each condition studied (three independent experiments were performed for each condition).



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  		Figure 4. Electrophoretic separation of immunoprecipitated 3H-labeled peptides with anti-GHRH from pulse-chase experiments in hypothalamic neurons. The immunoprecipitates were electrophoresed on an SDS-polyacrylamide gel, and the counts were plotted against gel slice. The mol wt of the peaks are indicated based on the migration of mol wt standards. This figure represents a typical profile of three independent experiments. The molecular mass markers shown are 6.5 and 2.8 kDa.

 


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  		Figure 5. Electrophoretic separation of immunoprecipitated 35S-labeled peptides with anti-GHRH from pulse-chase experiments in hypothalamic neurons. The immunoprecipitates were electrophoresed on a SDS-polyacrylamide gel, and the counts were plotted against gel slice. The mol wt of the peaks are indicated based on the migration of mol wt standards. This figure represents a typical profile of three independent experiments. The molecular mass markers shown are 6.5 and 2.8 kDa.

 


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  		Figure 6. Electrophoretic separation of immunoprecipitated 3H-labeled peptides with anti-GHRH-RP from pulse-chase experiments in hypothalamic neurons. The immunoprecipitates were electrophoresed on a SDS-polyacrylamide gel, and the counts were plotted against gel slice. The mol wt of the peaks are indicated based on the migration of mol wt standards. This figure represents a typical profile of three independent experiments. The molecular mass markers shown are 6.5 and 2.8 kDa.

 


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  		Figure 7. Electrophoretic separation of immunoprecipitated 35S-labeled peptides with anti-GHRH-RP from pulse-chase experiments in hypothalamic neurons. The immunoprecipitates were electrophoresed on a SDS-polyacrylamide gel, and the counts were plotted against gel slice. The mol wt of the peaks are indicated based on the migration of mol wt standards. This figure represents a typical profile of three independent experiments. The molecular mass markers are 6.5 and 2.8 kDa.

 
Figure 4AGo shows a pulse-chase experiment from cells initially pulsed for 30 min with 700 µCi [3H]leucine followed by chase times of 30, 60, and 90 min with an excess of cold leucine. The extracted peptides were immunoprecipitated with anti-GHRH and followed by fractionation on SDS-PAGE, slicing, and counting. At time zero, only a 10.5-kDa peptide similar in size to the pro-GHRH precursor was observed (31, 32). After 30 min of chase, approximately 50% of the precursor was processed, and a new intermediate form of about 8.8 kDa was generated. In addition, a 5.2-kDa peptide, which had the same electrophoretic mobility as synthetic iodinated GHRH [prepro-GHRH-(31–73)], and a smaller form of about 3.5 kDa were observed (Fig. 4AGo). Based on stoichiometric calculations, approximately 50% of the precursor (9 Leu) was converted to the 8.8-kDa peptide (8 Leu) and the 5.2-kDa peptide (4 Leu). After 60 min of chase (Fig. 4BGo), 75% of the precursor was proteolytically processed. The 8.8-kDa intermediate peptide was processed to smaller forms that did not accumulate within the cell. We propose that the 8.8-kDa intermediate represents the prepro-GHRH-(31–104) sequence, which is produced after initial cleavage at the basic amino acids at positions Arg29-Arg30 from the N-terminal side of the precursor. A second proteolytic cleavage at the single Arg74 residue at the amino acid motif of Arg71-Phe72-Asn73-Arg74{downarrow}His75-Leu76 results in a 5.2-kDa peptide that corresponds to GHRH, as supported by the similar mobility of synthetic GHRH and the previously described C-terminal sequence (31, 32). This cleavage fits with the proposed cleavage sites of type III precursor proteins, which are cleaved by the family of prohormone convertases at single basic residues following the formula: (Basic)-X-(Basic)-X-(Basic)-X-X-R {downarrow}X-X (33).

At 30 min chase time, a 3.5-kDa moiety was detected. This peptide could be generated by one of two mechanisms (Fig. 9Go): 1) cleavage at the Arg64 located within the amino acid motif: Arg59-Gln60-Gln61-Gly62-Glu63-Arg64{downarrow}Asn65-Gln66 to generate prepro-GHRH-(31–63) (~3.5 kDa); this motif is also characteristic of the type III precursor that serves as substrate for the prohormone convertases; and 2) cleavage at the Arg41-Arg42 located within the amino acid motif: Thr37-Ser38-Ser39-Tyr40-Arg41-Arg42{downarrow}Ile43-Leu44 to generate prepro-GHRH-(43–73) (~3.5 kDa). This motif is characteristic of the type II precursor that serves as substrate for the prohormone convertases. After 60 min of chase (Fig. 4BGo), the 10.5-kDa peptide was substantially processed, as the 8.8-kDa peptide was still being generated. At 90 min of chase, no visible peptides were detected. These data suggest that after 60 min, the pro-GHRH molecule is rapidly and completely processed to its smaller forms. However, at longer chase times, no GHRH was detected within the cell, suggesting that it is released to the medium (see Fig. 5BGo).



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  		Figure 9. Prepro-GHRH sequence indicating the sites of cleavage (arrows) to generate GHRH and GHRH-RP peptides. Further potential cleavages in the GHRH and GHRH-RP molecules are indicated by arrows. Basic residues are indicated by underlines. Leucine and methionine molecules are indicated in boldface.

 
Pulse-chase studies using 700 µCi [35S]methionine as radiolabeled isotope were performed to confirm the above findings (Fig. 5Go). After 30 min of chase, approximately 50% of the precursor was processed into 8.8- and 5.2-kDa peptides (Fig. 5AGo). Based on stoichiometric calculations at 30 min of chase, 50% of the processed precursor was distributed between the 8.8- and 5.2-kDa peptides. At 60 min of chase, almost complete processing of the precursor was observed, and a 3.5-kDa peptide accumulated. However, less of this 3.5-kDa peptide accumulated than would be expected based on the amount of original radiolabeled precursor. These results indicate that either the 3.5-kDa GHRH is quickly degraded, or it is released into the medium. To address this question, we analyzed the presence of peptides in the release medium from the same pulse-chase cultures. Only two moieties (5.2 and 3.5 kDa) were found in the release medium during the entire 60 min of chase (Fig. 5BGo). We hypothesize that the larger peptide (which has the same mobility as the iodinated synthetic GHRH) is GHRH, and the smaller one is a truncated form of GHRH produced after further cleavage. Interestingly, more of the 3.5-kDa peptide than of the 5.2-kDa peptide was present in the release medium.

The ICC and IEM experiments presented in Figs. 2Go and 3Go indicate storage of immunoreactive GHRH in SGs in hypothalamic neurites in culture. However, the pulse-chase experiments presented in Fig. 4Go, A and B, indicate that after 60 min of chase (Fig. 4BGo), 75% of the precursor is proteolytically processed, and smaller forms do not accumulate within the cell. As the immunohistochemical data suggest that GHRH is released through the regulated pathway, but the pulse-chase studies raise the possibility that GHRH is released through the constitutive pathway, we sought to clarify this issue. After a 45-min pulse-chase, cells were treated with either PMA or medium for an additional 45 min. The results depicted in Fig. 4CGo demonstrate that cells subjected to PMA stimulation release substantially more GHRH into the release medium than controls, indicating that GHRH is stored in SGs and is released through the regulated pathway.

To further characterize pro-GHRH processing, GHRH-RP antibodies were used to detect processing products from similar pulse-chase experiments with [3H]leucine and [35S]methionine (Figs. 6Go and 7Go). In Fig. 6AGo, at time zero chase, only the 10.5-kDa peptide was present. After 30 min of chase, an 8.8-kDa form was generated, as seen using GHRH antibodies. In addition, peptides of 3.6 and about 2.2 kDa were produced. The 3.6-kDa peptide had the same electrophoretic mobility as the iodinated synthetic GHRH-RP peptide. We hypothesize that this peptide is derived from the proteolytic cleavage at Arg74 described above, which results in both GHRH and GHRH-RP. Similar to the profile observed in Fig. 5BGo, Fig. 6BGo shows that at 60 min, there was accumulation of the 8.8- and 3.6-kDa peptides, but by 90 min of chase, the smaller forms were not detected. Moreover, when the cells were radiolabeled with [35S]methionine (Fig. 7Go, A and B), a similar profile of processing (Fig. 6Go) was found, resulting in the generation of an 8.8-kDa intermediate form and a 3.6-kDa peptide. In these experiments, we also observed that end products of processing accumulated in the release medium, but not within the cell.

Regarding the formation of the approximately 2.2-kDa peptide, the results from Figs. 6Go and 7Go suggest that this peptide derived from a further proteolytic cleavage of the GHRH-RP peptide. As GHRH-RP contains several monobasic residues, we do not know which enzymatic cleavage generates the 2.2-kDa peptide. However, we propose that it is prepro-GHRH-(75–92), because, as demonstrated below, this peptide stimulates cAMP and has a molecular mass of about 2.2 kDa. Therefore, we propose that GHRH-RP is further processed at Leu92, which is located within the amino acid motif Glu-Ser90-Ile91-Leu92{downarrow}Gln93-Gly94-Phe95-Pro96-Arg97.

Activation of the PKA pathway by novel peptides derived from pro-GHRH processing
Based on pulse-chase analysis, several peptides were synthesized and tested for potential biological action in a transient transfection system that detects cAMP activation (34, 35). In these studies, GHRH-RP and GHRH stimulated PKA-dependent luciferase activity 2.6- and 4-fold, respectively, above that in vehicle-treated control cells. The results from a representative experiment are presented in Fig. 8Go. Interestingly, prepro-GHRH-(75–92)-NH2 significantly increased cAMP activity 6-fold (P < 0.05), similar to that observed with forskolin treatment. Prepro-GHRH-(75–83) treatment caused no change in cAMP. As expected, PMA had no effect.



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  		Figure 8. cAMP activation by pro-GHRH-processing products. Sertoli cells were transfected with the PathDetect cAMP response element-binding protein trans-reporting system for detection of cAMP stimulation. Luciferase activity from vehicle-treated cells (v) was compared with that from cells treated with GHRH (100 nM), GHRH-RP (100 nM), prepro-GHRH-(74–83) (100 nM), prepro-GHRH-(74–92) (100 nM), forskolin (FSK; positive control), or PMA (negative control). Cells transfected with pFR-luc served as the vector control, and pFC-pka was the internal positive control. Results represent one of at least four individual experiments containing three transfections per treatment (mean ± SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Posttranslational processing of neuropeptides and prohormones, usually at pairs of basic and, less frequently, at monobasic residues is essential for maturation of these peptides (36, 37). In this study we propose for the first time a model of posttranslational processing for the GHRH precursor and suggest that more than one biologically important peptide results from this process. Multiple approaches were used to biochemically and anatomically identify the localization of pro-GHRH and its processing products in adult rat hypothalamus and in primary cultures of hypothalamic neurons. Identification of the GHRH precursor was demonstrated by immunoprecipitation of radiolabeled peptides extracted from hypothalamic neurons in culture. The localization of immunoreactive pro-GHRH-derived peptides was demonstrated by fluorescence ICC in rat hypothalamus and neurons in culture and by IEM. Pro-GHRH biosynthesis and processing were demonstrated by pulse-chase experiments using [3H]leucine and [35S]methionine as isotope markers.

Using specific GHRH-RP antisera, immuonoactivity was localized to nerve terminals and fibers in the median eminence, but not in any other areas of the brain. As the antisera also recognize other intermediate forms (see pulse-chase experiments), this study does not necessarily indicate that mature GHRH-RP is localized to the median eminence. However, posttranslational processing of other prohormones during their vectorial transport from the trans-Golgi network to mature SGs results in only mature peptides reaching the median eminence (38). In the median eminence, peptides are further transported to the pituitary through the portal vessels. The cell bodies and neurites from individual neurons were stained positively for both GHRH and GHRH-RP.

After cloning the corresponding complementary DNAs, it was determined that the GHRH sequence is derived from the prepro-GHRH-(1–104) precursor after removal of the signal leader sequence peptide, followed by two proteolytic cleavages, one at the N- and the other at the C-terminal regions of prepro-GHRH-(30–74). This reaction generates a biologically active GHRH [prepro-GHRH-(31–73)] after removal of the basic residues (32, 39). Our pulse-chase experiments, using both [35S]methionine and [3H]leucine, provide substantial support for this hypothesis and further demonstrate the order by which the end products of processing are generated. These studies also suggest that more than 50% of the original precursor is processed within 30 min after its biosynthesis to give rise to an 8.8-kDa intermediate form, GHRH, GHRH-RP, and four smaller peptides. These small peptides derive from further cleavage of the 5.2-kDa GHRH peptide and the 3.6-kDa GHRH-RP peptide, possibly functioning as either type II or type III precursors. Before this report, these smaller moieties had not been identified. We also propose that the 8.8-kDa form detected in the first stages of processing is derived from an N-terminal initial cleavage at the Arg30 of the original prepro-GHRH precursor, as originally proposed to be the N-terminal end of GHRH (32, 39). A remaining N-terminal prepro-GHRH-(20–30) peptide of about 1.1 kDa may also be formed after the generation of the 8.8-kDa intermediate. We have yet to confirm this hypothesis, because we do not currently have an antibody against this sequence. Figures 9Go and 10Go depict the proposed model of pro-GHRH processing based on the described pulse-chase experiments.



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  		Figure 10. Proposed model of pro-GHRH processing to its end products.

 
In humans, proteolytic processing of the pro-GHRH precursor peptide involves cleavage of the signal peptide [prepro-GHRH-(1–19)] followed by two subsequent cleavages at the N- and C-terminal sides of GHRH to form a 44-amino acid peptide [prepro-GHRH-(31–74)] (39, 40). These proteolytic cleavages occur at the pair of basic residues Arg29-Arg30 and at the monobasic Arg73 site (39, 41). This reaction is followed by a subsequent amidation at the terminal glycine (Gly73) residue, which acts as an amide donor to amino acid 72 (leucine) (42). The final amidated product [prepro-GHRH-(31–73)NH2] is the major GHRH form produced by the human hypothalamus. Two other nonamidated GHRH peptides have been identified in human tissues, a 40-amino acid GHRH [prepro-GHRH-(31–70)-OH] and a 37-amino acid GHRH [prepro-GHRH-(31–67)-OH] (39, 43). Although, in general, processed peptides are flanked by paired basic amino acids (33), cleavage has been reported at sites containing a single arginine, such as in the processing of arginine vasopressin-neurophysin (44) and ACTH from POMC (45). The alternative GHRH products, prepro-GHRH-(31–67)-OH and prepro-GHRH-(31–70)-OH represent cleavage at other single arginine sites and are most likely natural variants. The forms prepro-GHRH-(31–73)-OH and prepro-GHRH-(31–70)-OH have been identified in the hypothalamus and placenta, whereas prepro-GHRH-(31–67)-OH has only been identified in a GHRH-secreting pancreatic tumor (12, 13, 46). All three GHRH forms have similar bioactivity in the release of pituitary GH.

GHRH has been characterized in many species (46). The amino-terminal end of the GHRH peptide is most conserved across species, with the greatest similarity to human GHRH seen in the larger mammals (e.g. porcine, ovine, caprine, and bovine). The biological activity of GHRH is influenced predominantly by the more conserved amino-terminal residues, with loss of the first two amino acids rendering the peptide inactive (46, 47). Although loss of carboxyl-terminal amino acids results in some loss of function, synthetic prepro-GHRH-(31–59) retains most of the GH-releasing potential of the natural prepro-GHRH-(31–73) form (46, 47). We propose that the smaller forms of GHRH observed in our pulse-chase studies may be similar to the shorter forms of GHRH found in human tissue.

Unlike larger mammals, rodent and human GHRH gene products demonstrate greater differences (31, 32, 46). The smaller GHRH peptides in rat and mouse contain 43 and 42 amino acid residues, respectively, with approximately 70% homology between rodent and human GHRH (31, 32). Similar to other species, the homology is greatest in the amino-terminal residues. As no amide donor is present at the carboxyl-terminus of the GHRH peptide, both rat and mouse GHRH are nonamidated (31, 32). In larger mammals, including humans, amidation of the GHRH peptide and probably also of the carboxyl-terminal peptide (GHRH-RP) occurs, a posttranslational modification commonly associated with biological function (43, 46). Although amidation does not occur in rat and mouse GHRH, synthetic rat prepro-GHRH-(31–59) can become functional by C-terminal amidation (48). Similarly, human GHRH biological activity can be retained in the C-terminal shortened 31–59 synthetic form by amidation.

The data presented in Figs. 4–7GoGoGoGo suggest that during pro-GHRH processing, multiple peptides are generated, in addition to GHRH. It is important to emphasize that a proportion of those four end products: GHRH, a 3.5-kDa GHRH peptide, GHRH-RP, and a 2.2-kDa GHRH-RP peptide were released into the medium. This indicates that complete intracellular degradation of these peptides does not occur by lysosomal or other degradation enzymes. Support for the production of multiple end products from pro-GHRH biochemical processing also comes from work of Vazquez (49) in which rat hypothalamic cells were cultured in the presence of [3H]arginine. These investigators identified four major peaks by HPLC, one of which coeluted with GHRH. The other three peptide peaks in their study could correspond to the products identified by our experiments. In other studies done in human hypothalamus, three major peptides were found (50): one with a molecular mass of 30–45 kDa (9.99 pg/g wet tissue), a second peptide of 10 kDa (29.37 pg/g wet tissue), which resembles the size of the pro-GHRH precursor; and a third major peptide of 5 kDa (corresponding to synthetic human pro-GHRH-(31–74)-NH2; 197.67 pg/g wet tissue).

In an attempt to clarify whether GHRH is released through the consititutive or regulated secretory pathway, we performed additional experiments with PMA as a secretagogue. PMA treatment results in notable GHRH secretion, thus supporting the concept that GHRH is released via the regulated secretory pathway. However, we also observed peptide release into the medium even without stimulation. This could be due to stimulatory factors in the culture medium or, alternatively, the production of endogenous stimuli by the neuronal cultures known to contain an array of different neuronal types (25, 26). It is interesting to note that, similar to our study, Vazquez and colleagues found that most of the GHRH peptide they identified was present in the release medium and not in the cell content. Even though previous studies in human hypothalamus demonstrate the presence of a 5-kDa peptide (see above) as the main small moiety produced, characterization of the mature peptides produced in the rat has not yet been clearly determined. Thus, it is possible that in rodents, a shorter form of GHRH is primary stimulus of GH release.

Cultured hypothalamic neurons derived from dissociated fetal rat diencephalon have provided a valuable system for studying the mechanisms of neuropeptide processing and secretion (25, 51, 52, 53). These cells develop a complex dendritic and axonal network, as indicated by staining with specific antibodies against microtubulin-associated protein, neurofilaments, and {gamma}-aminobutyric acid (51). In addition, hypothalamic neurons expressing neuropeptide Y messenger RNA and its peptide, as well as opioid-like peptides, vasopressin, substance P, and neurotensin, have been documented (51).

In the current study, we have shown that three peptides derived from pro-GHRH processing, GHRH, GHRH-RP, and prepro-GHRH-(75–92)-NH2, stimulate cAMP in transfected Sertoli cells. This along with preliminary data demonstrating the in vitro and/or in vivo stimulation of SCF by these peptides imply that they have functions beyond the GH-stimulating activity of GHRH.

In summary, we present here for the first time a model of the biochemical processing of pro-GHRH. In this work we have identified six peptide products derived from the pro-GHRH precursor, several of which probably have distinct biological activity. We speculate that this processing is mainly dependent on the action of proteolytic converting enzymes, including PC1, PC2, PC5, and possibly furin, but may also involve thiol (54) and aspartyl proteases (55). Further studies using cotransfections of pro-GHRH and members of the prohormone convertase complementary DNAs are needed to confirm the exact amino acid sequence site where the processing of pro-GHRH occurs. This will also permit the generation of larger amounts of material for sequencing analysis and the opportunity to characterize which prohormone convertases are used to fully process pro-GHRH.


   Acknowledgments
 
The authors thank Paul Breyer, M.D., for his work on the GHRH-RP IHC, and April R. Bartnick and James Rothrock for their excellent technical assistance.


   Footnotes
 
1 This work was supported by NIH Grant RO1-HD-34789. Back

Received March 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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