Characterization of Pro-ACTH/Endorphin-Derived Peptides in Rat Hypothalamus

Home , Endorphins, Lipotropin, Methionine

The Journal of Neuroscience March 1966, 6(3): 637-649

Ronald B. Emeson and Betty A. Eipper Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The proteolytic processing pattern of pro-ACTH/endorphin in Browne et al., 198 la, b; Douglass et al., 1984; Eipper and Mains, rat hypothalamus is similar to the pattern in the pars interme- 1980, 1982; Krieger et al., 1980; Rudman et al., 1979; Seidah dia; peptides the size of 8-endorphin, */-lipotropin (v-LPH), cor- and Chretien, 198 1; Zakarian and Smyth, 1982a, b). ticotropin-like intermediate lobe peptide (CLIP), a-melanotro- Extensive immunohistochemical mapping studies utilizing pin (?I-MSH), joining peptide, and glycosylated r,-MSH all antisera raised against several pituitary ACTH/endorphin-de- represent predominant end products. Equimolar amounts of rived peptides have characterized a large number of neuronal &endorphin-, a-MSH-, CLIP-, T-LPH-, and joining peptide- pathways containing antigenic determinants of the ACTH/en- related immunoreactivity are found in hypothalamic extracts dorphin system throughout the mammalian brain (Bloom et al., (-3 pmol per hypothalamus). Although the proteolytic process- 1980; Joseph, 1980; Oliver and Porter, 1978; Pelletier, 1979; ing pattern in the hypothalamus is similar to that in the pars Schwartzberg and Nakane, 1982; Watson et al., 1978; Zim- intermedia, a tissue-specific posttranslational processing pat- merman et al., 1978). Although immunoreactive fibers are found tern was detected. Ion-exchange analysis of &endorphin-sized in many regions of the forebrain and brain stem, there is general immunoreactive material from hypothalamic extracts resolves agreement that positively staining cell bodies are located exclu- three major forms, corresponding to &endorphin(l-31), &en- sively in the arcuate nucleus of the hypothalamus and the dorsal dorphin( l-27), and &endorphin( 1-26). The a-iV-acetylated caudal medulla in the region of the nucleus of the solitary tract forms of endorphin represent less than 10% of the total /?-en- (Khachaturian et al., 1983; Romagnano and Joseph, 1983; dorphin immunoreactivity. Analyses of hypothalamic a-MSH- Schwartzberg and Nakane, 1983). sized molecules with acetyl- and amide-directed a-MSH anti- The structure of the single gene that codes for pro-ACTH/ sera suggest that hypothalamic a-MSH is fully amidated, but endorphin within the rat genome (Drouin and Goodman, 1980; largely not a-l\r-acetylated. Fractionation by reverse-phase high- Drouin et al., 1983) suggests that any differences that exist be- performance liquid chromatography (HPLC) confirms that tween ACTH/endorphin-derived peptides from multiple tissues > 85% of the a-MSH immunoreactivity corresponds to ACTH( l- are the result of tissue-specific posttranslational processing rath- 13)NH, or its sulfoxide, and less than 10% corresponds to er than the result of expression of two different genes. Recent a-MSH [a-N-acetyl-ACTH(l-13)NH,] or its sulfoxide. Isoelec- studies have demonstrated that, at the level of gel filtration, the tric focusing demonstrates that 83-93% of hypothalamic CLIP processing of pro-ACTH/endorphin-derived peptides in rat hy- is phosphorylated. Isoelectric focusing suggests that the major- pothalamus more closely resembles the pattern for proteolytic ity of the hypothalamic y-LPH-sized immunoreactive material processing in the pars intermedia than in the pars distalis, with is indistinguishable from */-LPH synthesized by pituitary me- both a-melanotropin (a-MSH) and @-endorphin-sized material lanotropes. The minor extent of a-iV-acetylation of a-MSH and representing the predominant end products (Bamea et al., 1982; &endorphin, the limited carboxyl-terminal proteolysis of &en- Dennis et al., 1983a; Gramsch et al., 1980; Loh et al., 1980; dorphin, and the extensive phosphorylation of CLIP represent Orwoll et al., 1979); a summary of the nomenclature for pro- major differences between the posttranslational processing pat- ACTH/endorphin-derived peptides is shown in Figure 9. terns of pro-ACTH/endorphin in the hypothalamus and pars In the intermediate lobe of the pituitary, a-MSH-like material intermedia. has been shown to exist in three distinct forms differing in their degree of acetylation. Although a-N,O-diacetyl-a-MSH repre- Adrenocorticotropin (ACTH) and P-lipotropin (P-LPH) are syn- sents the major form of a-MSH synthesized by rat pituitary thesized as parts of a larger common precursor molecule [pro- melanotropes (Browne et al., 1981a; Glembotski, 1982; Rud- ACTH/endorphin or pro-opiomelanocortin (POMC)], which is man et al., 1979), there is disagreement over the acetylation posttranslationally processed, in a tissue-specific manner, to yield state of hypothalamic a-MSH-like material (Evans et al., 1982; differing end products in the anterior and intermediate lobes of Geis et al., 1984; Loh et al., 1980; O’Donohue et al., 1981). the pituitary. The co- and posttranslational modifications in- Previous studies have shown that the major forms of ,&en- volved in the processing of this 30 kDa precursor protein in- dorphin in the rat intermediate pituitary are the a-N-acetylated clude defined and ordered proteolysis, glycosylation, phospho- derivatives of &endorphin( l-26) and &endorphin( l-27) rylation, a-N-acetylation and a-amidation (Bennett et al., 1982; whereas the major form produced by the anterior lobe corticotropes is ,&endorphin( l-3 1) (Akil et al., 198 1; Bennett et al., Received May 20, 1985; revised Aug. 12, 1985; accepted Aug. 23, 1985. 1983; Eipper and Mains, 1981; Weber et al., 1981; Zakarian We are grateful to Henry Keutmann for quantitative amino acid analyses of and Smyth, 1982a, b). There is still considerable controversy, purified rat y,-MSH and joining peptide, and to Dick Mains for many enlightening however, over the extent of a-N-acetylation (Weber et al., 198 1; scientific discussions and critical reading of the manuscript. This work was sup- Zakarian and Smyth, 1982a, b) and carboxyl-terminal proteol- ported by DA-00266, DA-00098, and the M&night Foundation. ysis (Dennis et al., 1983a, b; Liotta et al., 1984; Zakarian and Correspondence should be addressed to Ronald B. Emeson, Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe Smyth, 1982a, b) of B-endorphin in the rat brain. Street, Baltimore, MD 21205. Previous studies on the posttranslational processing of cor- Copyright 0 1986 Society for Neuroscience 0270-6474/86/030837-13$02.00/O ticotropin-like intermediate lobe peptide (CLIP) by rat pituitary

837 838 Emeson and Eipper Vol. 6, No. 3, Mar. 1986

melanotropeshave demonstratedthat, in addition to the proteolytic cleavagesthat produce CLIP from ACTH, there are Radioimmunoassays further modifications to which CLIP is subjected, including Radioimmunoassays for P-endorphin utilized antiserum Danielle [spe- phosphorylation, N-linked glycosylation, and removal of the cific for fl-endorphin( 10-19); Mains and Eipper, 19791. Radioimmu- carboxyl-terminal phenylalanine residue (Bennett et al., 1982; noassays for a-MSH utilized antiserum Ann [a general ACTH( 1-13) Eipper and Mains, 1982). Previous studieson the biochemical assay; Glembotski, 19821, antiserum Annette [an amide-directed a-MSH assay; Eipper et al., 19831, and antiserum Patti [an acetyl-directed (Y- characterization of hypothalamic CLIP have demonstratedmul- MSH assay; Eipper et al., 19831. Radioimmunoassays for CLIP tiple peaks of CLIP-related immunoreactivity upon reverse- [ACTH( 18139)] utilized antiserum -Kathy [specific for ACTH(34-39); phase(RP) HPLC analysis,suggesting the occurrence of post- Mains and EiDDer. 19831. Antiserum Kathy showed less than 0.2% M translational modificationssimilar to thoseoccurring in the pars cross-reactivitywith ACTH(7-38) compared with either hACTH( l-39) intermedia(Liotta and Krieger, 1983a;Smith et al., 1982; Turner or ACTH( 18-39), demonstrating the necessity of the carboxyl-terminal et al., 1983). phenylalanine residue for full cross-reactivity. Radioimmunoassays for The demonstration of ACTH/endorphin-derived molecules y-LPH utilized antiserum Henrietta; as reported previously for rat pi- in multiple tissues,with the potential of posttranslationalmod- tuitary (Eipper and Mains, 1979), immunoassays for rat hypothalamic ification into products both similar to and different from those r-LPH-related peptides utilizing mouse /3-lipotropin as a radiolabeled trace and standard showed nonparallel displacement. elaboratedby the anterior and intermediate pituitary lobes, has In order to quantitate the relative levels of y-LPH-related molecules raised a number of fundamental questionswith regard to the in gel filtration fractions, the amount of each fraction to be assayed was chemical nature, biosynthetic pathway, regulation, and func- varied so that the actual amount of ‘251-&,,-LPH (mouse @-lipotropin) tion(s) of thesepeptide products in the different tissuesin which displaced by each sample was similar. All assays were performed as they can be found. Since the various peptide products derived previously described. from a singleprecursor molecule are releasedin a coordinated Radioimmunoassays for y-MSH-related peptides were performed with fashion (Liston and Rossier, 1984; Mains and Eipper, 1979; a 1:800 dilution of antiserum Gertrude (Eipper et al., 1983), using Tager et al., 1975), and each may interact independently with purified AtT-20 16K fragment (Keutmann et al., 1979) as a radiolabeled an appropriate target tissue or modulate the effectsof another trace and purified rat intermediate pituitary n-MSH as a standard. Assays were performed in 50 mM sodium phosphate, pH 7.6, containing peptide product with such a target tissue (Farese et al., 1983; 0.025% Triton X- 100; the final assay volume was 200 ~1, and incuba- O’Donohue et al., 1981; Pedersenet al., 1980; Scheller et al., tions were carried out for 18 hr at 4°C with approximately 10,000 cpm 1984; Walker et al., 1980) a coordinated study on the quanti- of 12jI-labeled peptide per tube. Double-antibody immunoprecipitation tation and biochemical characterization of hypothalamic pro- was used to separate bound from free peptide. Under these conditions, ACTH/endorphin-derived peptides was undertaken. the assay had a midpoint of approximately 200 fmol; maximum net binding was 25%, and nonspecific binding was 0.5%. Antiserum Ger- trude demonstrated approximately 70% molar cross-reactivity with pu- Materials and Methods rified AtT-20 16K fragment and less than 0.01% molar cross-reactivity Tissueextraction and samplepreparation with cu-MSH, CLIP, purified mouse fl-LPH, purified rat joining peptide, Adult male Sprague-Dawley rats (125-250 gm) were used for all ex- human ACTH, and camel fl-endorphin. periments. The animals were decapitated and the whole brains were Radioimmunoassays for amidated joining peptide were performed rapidly removed. Hypothalamic tissue (- 25 mg per hypothalamus) was with a 1:4000 dilution of antiserum Jamie; the antiserum was generated excised and homogenized by hand in 5 volumes (wt/vol) of 1% trifluo- by injection of a female New Zealand White rabbit with a modified syntheticcarboxyl-terminal fragment ofrat-joining peptide coupled with roacetic acid, 1 M HCl, 5% formic acid, and 1% NaCl (Bennett et al., 1978) by means of a conical glass homogenizer with ground-glass pestle. glutaraldehvde to BSA ID-Tyr-Pro-Glu-Pro-Ser-Pro-Am-Glu-NH, or The boundaries of the hypothalamic fragment extended from the an- D-Tyr-joining peptide( 12-l 8)NH,; Vega Biotechnologiesj. Assays were terior margin of the preoptic area to the anterior border of the mam- oerformed as described. using D-Tvr-ioinina nentide(l2-18)NH, as millary bodies. Extracts were frozen and thawed twice and centrifuged standard and trace (Eipper and-Mains, unpubisheh observations). The at 1500 x g for 15 min at 4°C to remove insoluble material. The joining peptide assay demonstrated approximately 70% molar cross- supematants were removed and the pellets were reextracted by resus- reactivity with purified rat-joining peptide and less than 0.01% molar pension in l-2 ml TFA/HCl/HCOOH/NaCl and then centrifugation; cross-reactivity with joining peptide( 12-l 9) (Vega Biotechnologies), & the supematants were pooled. endorphin, (u-MSH, ACTH, CLIP, purified rat y,-MSH, purified mouse Tissue extracts were cycled five times onto a C,, Sep-Pak cartridge P-LPH, and equine @-MSH. (Waters Associates), washed with 20 ml 0.1% trifluoroacetic acid and eluted with 4 ml 80% acetonitrile in 0.1% trifluoroacetic acid (Bennett RP-HPLC analysis of cr-MSH-related material et al., 198 1b); 1 mg BSA was added to the Sep-Pak eluate as a carrier a-MSH-sized material obtained by chromatography on Sephadex G-75 protein. The tissue extracts were dried under reduced pressure after or gel filtration HPLC was dried under reduced pressure, resuspended addition of 2-mercaptoethanol (l%). in 0.1% trifluoroacetic acid, and analyzed on a C,, PBondapak column (Waters Associates; 4 x 250 mm) using the triAuoroacetic~acid/aceto- Gel filtration nitrile system of Bennett et al. (198 1b); specific elution conditions are For gel filtration using Sephadex G-75 (40-120 pm), tissue extracts were indicated in the figure legends. Synthetic marker peptides were pur- redissolved in 50% acetic acid containing 5% 2-mercaptoethanol; the chased from Bachem. The elution positions of synthetic standard pep- Sephadex G-75 column (0.9 x 58 cm) was eluted with 10% formic acid, tides were determined in parallel RP-HPLC analyses by monitoring 20 &ml bovine serum albumin, and 0. II 2-mercaptoethanol (Eipper absorbance at 220 nm. and Mains, 1982; Glembotski, 1982). For HPLC gel filtration, tissue extracts were redissolved in 250 ~1 of 32% acetonitrile containing 0.1% Ion-exchange analysisof /3-endorphin-relatedmaterial trifluoroacetic acid; the samples were applied to a Bio-Sil TSK-400 P-Endorphin-sized material obtained by chromatography on Sephadex column (7.5 x 300 mm) connected in series to a Bio-Sil TSK-250 (7.5 x G-75 or gel filtration HPLC was dried under reduced pressure and 300 mm) and a Bio-Sil TSK-125 (7.5 x 300 mm) column (Bio-Rad). analyzed by ion-exchange chromatography on Sulfopropyl-Sephadex The columns were eluted with 32% acetonitrile in 0.1% trifluoroacetic (SP-Sephadex-C25- 120), as previously described (Mains and Eipper, acid (Bennett et al., 198 1b; Eipper et al., 1983) at a flow rate of 1 ml/ 198 1; Zakarian and Smyth, 1979). A trace amount of ‘H-labeled j3- min. The elution times of several standard proteins and synthetic pep- endorphin( l-3 1) obtained from AtT-20 cells (Mains and Eipper, 198 1) tides were determined by monitoring absorbance at 220 nm. Recoveries, was included in each sample as an internal marker. For immunoassays, determined bv the use of various )H-labeled pro-ACTH/endoruhin- fractions were dried under reduced pressure and dissolved in 0.1 M Tris- derived peptihes purified from primary cultures of rat intermediate HCl [Tris(hydroxymethyl)aminomethane] containing 2 mg/ml BSA, pH pituitary or mouse corticotropic tumor cells (Keutmann et al., 1981), 8.0. were in excess of 90%. Whole hypothalamic tissue extracts or ,f3-endorphin-sized material The Journal of Neuroscience Rat Hypothalamic Pro-ACTH/Endorphin-Derived Peptides 839

from hypothalamic extracts were dried under reduced pressure, redissolved with 10 mM ammonium formate, pH 2.5, in 30% acetonitrile, and analyzed by ion-exchange HPLC on a Bio-Sil TSK CM-2-SW cation-exchange column (4.6 x 250 mm; Bio-Rad) equilibrated with 35 mM ammonium formate, pH 2.5, in 30% acetonitrile. Samples were eluted with a 60 min linear gradient to 326 mM ammonium formate, pH 2.5, in 30% acetonitrile; the flow rate was 0.7 ml/min. Buffers were prepared from a stock solution of 5 M formic acid titrated to pH 2.5 with ammonium hydroxide. The elution positions of six synthetic p-endorphin standards (Peninsula) were determined by monitoring absorbance at 280 nm (Fig. 1). As shown in Figure 1, the @-endorphin-related peptides are eluted primarily on the basis of their charge at pH 2.5 (charge given in brackets): acetyl-&endorphin( l-26) [ + 31, &endorphin(l-26) [+4], acetyl-&endorphin(l-27) [+4], @,-endorphin(l-27) ]+51. ace&l-b-endorphiml-31) 1+61, and&endomhin(l-31) 1+71. The fact-that acetyl-&-endorphin(l-27)-]+4] and &-endorphin(i-26) [+4] are separated by this fractionation procedure indicates that separation is not based solely on charge. Camel and rat P-endorphin(l-31) have .J::::::::::::::..::::...... ::::::...:,.:::::...... o identical amino acid sequences (Bennett et al., 1983; Drouin and Good- 0 5 10 IS 20 25 30 35 40 45 50 55 60 w man, 1980; Li and Chung, 1976); fl-endorphin(l-26) is identical in Retention time (mln) humans, rat, and camel (Chang et al., 1980). Application of trace amounts Figure 1. Ion-exchange HPLC of synthetic @-endorphin-related pep- of ‘H-labeled AtT-20 &endorphin( l-3 1) (co.05 pmol) to this ion-ex- tides. Fifteen micrograms of synthetic (I) acetyl-&-endorphin( l-26), change system, in the absence oftissue extract or carrier protein, resulted (2) acetyl-&-endorphin(l-27), (3) 8,-endorphiml-26), (4) &-endor- in recovery of only 46% of the radioactivity. Inclusion of 2 ng (0.6 pmol) phin( l-27), (5) acetyl-&-endorphin( l-3 l), and (6) &endorphin( l-3 1) of synthetic &endorphin( l-3 1) along with the ‘H-labeled sample in- were mixed and then separated on a Bio-Sil TSK CM-2-SW cation- creased recovery of radioactivity to levels in excess of 80%. In analyses exchange column as described in Materials and Methods. The retention of hypothalamic extracts, recoveries of j3-endorphin-related immuno- times of the standards were determined by monitoring absorbance at reactivity were greater than 70%. 280 nm. The identities of the peaks were determined by a series of Oxidation of the methionine residue in @-endorphin can occur during analyses in which the peptides were chromatographed both separately the course of tissue extraction and subsequent biochemical analyses and together. (Houahton and Li. 1979: Mains and EiDDer. 1981). Thus. we assaved the ei?ect of methionine oxidation on eluiion of peptides from the ibn- be carried out subsequently, trypsin was inactivated by addition of exchange (IEX)-HPLC column. Synthetic &-endorphin( l-3 1) and ace- phenylmethylsulfonyl fluoride (0.3 mg/ml) and incubation at 37°C for tyl-&,-endorphin( l-26) were oxidized with hydrogen peroxide and ana- 15 min. lyzed on the IEX-HPLC column both separately and together with nonoxidized peptide standards. Although the elution positions of oxidized &-endorphin( l-3 1) and acetyl-&-endorphin( l-26) were earlier Results than those of their corresponding nonoxidized counterparts (data not Geljltration analysesof hypothalamic extracts shown), the altered retention times were not sufficiently different to At the level of gel filtration, the processingof pro-ACTH/en- interfere with peptide identification. dorphin in rat hypothalamus has previously been shown to re- semblethe pattern in the parsintermedia more closelythan the Oxidation of cu-MSH standards pattern in the pars distalis (Bamea et al., 1982; Dennis et al., Synthetic CZ-MSH [cY-N-acetyl-ACTH( l-l 3)NH,] and ACTH( l-l 3)NH, 1983a, b; Gramsch et al., 1980; Liotta and Kneger, 1983a, b; were suspended separately (1 mg/ml) in 1 M acetic acid to which 200 Loh et al., 1980). However, in none of these previous studies pi/ml of 30% hydrogen peroxide was added (O’Donohue et al., 1981). was there an attempt to quantitate and simultaneouslycorrelate The reaction was allowed to proceed for 30 min at room temperature, the processingof a number of the peptides derived from the and then an equal volume of 0.1% trifluoroacetic acid was added. The pro-ACTH/endorphin precursor, including (r-MSH, P-endor- reaction mix was then injected into the C,, PBondapak column, using the trifluoroacetic acid/acetonitrile system of Bennett et al. (1981b). phin, CLIP, r-LPH, r-MSH, and joining peptide. Hypothalamic Fractions containing methionine sulfoxide4-cY-MSH ([Met(O)]-ar-MSH) extracts were subjected to size fractionation utilizing Sephadex and [Met(O)]-ACTH( l-l 3)NH, were pooled, dried under reduced pres- G-75; fractions were analyzed by radioimmunoassaywith anti- sure. and dissolved in 1 mM HCl. seradirected againstseveral ACTH/endorphin antigenic determinants. Isoelectricfocusing Three peaksof amino-terminal ACTH-related immunoreac- y-Lipotropin and CLIP-sized immunoreactive material obtained by tivity were detected with a general ACTH( l-l 3) immunoassay chromatography on Sephadex G-75 were dried under reduced pressure; (Fig. 2, top; Table 1). The predominant end product was(u-MSH- samples were dissolved in 5 mM acetic acid containing 0.01% Triton sized. Two additional specieswith apparent molecular weights X-100 and applied to a 1 or 2 mm layer of Sephadex IEF resin (Phar- of 16,000 and 6500 Da representedless than 3 and 5% of the macia) containing 2% ampholytes (pH 2.5-5.0; Eipper and Mains, 1982). total ACTH-related immunoreactivity, respectively. ACTH- Electrophoresis was allowed to proceed for 16 to 20 hr at 600 V to sizedmaterial represented approximately 2% of the total ACTH- ensure focusing of peptides. Bands of resin (0.7 cm) were scraped from related immunoreactivity; while pro-ACTH/endorphin-sized the glass plate and eluted as indicated in the figure legends. Resin par- ticles were removed by eluting the resin in disposable columns (Ever- material was not detectable. green). Samples from one lane of the isoelectric focusing gel were sus- Three molecular weight forms of P-endorphin-related im- pended in 2 ml of water to determine the pH profile. munoreactivity were detected utilizing an antiserum directed against /3-endorphin(lO-19) (Fig. 2, top; Table 1). The P-en- Enzymatic digestions dorphin-sized material representedthe predominant end prod- Digestions with alkaline phosphatase were performed for 4.5 hr at 37°C uct, with minor amounts of both pro-ACTHIendorphin- and P-LPH-sized material (Fig. 2, top; inset). As can be seenin in 0.1 M NH,HCO, containina 0.5 me/ml BSA and 0.2 units/ml alkaline phosphatase(Sigma type III,42 U/mg); the reaction was stopped by Figure 2 (top), approximately equimolar amounts of ACTH- lyophilization (Eipper and Mains, 1982). Digestions with TPCK-trypsin and @-endorphin-relatedmaterial were found (3.4 f 0.3 pmol/ (Worthington, 236 U/mg) were carried out as previously described (Eip- hypothalamus; n = 6). per and Mains, 1977). When alkaline phosphatase digestions were to Two major peaks of carboxyl-terminal, ACTH-related im- 840 Emeson and Eipper Vol. 6, No. 3, Mar. 1966

pro-ACTH/mdorphln

-t’LPH CLIP I I

Figure 2. Gel filtration analysis of hypothalamic extracts. Tissue extracts from adult male rat hypothalami (17 to 48 hypothalami per extract) were fractionated on Sephadex G-75 as described in Materials and Methods. Ali- quots from 1 ml column fractions were analyzed by radioimmunoassay. Top, General amino-terminal ACTH-related peptides (Cl) and fi-endorphin-related peptides m. Middle, Carboxyl-terminal ACTH-related peptides (0) and r-LPH-related peptides (0). Bottom, Joining peptide-related molecules (A) and y-MSH-related peptides (A). Re- coveries for all peptides ranged between 84 and 117%. Data are expressed as fmol/fraction/hypothalamus. Arrows mark the elution positions of either ‘H- labeled peptides obtained from a mouse corticotropic tumor cell line (AtT-20) or primary cultures of rat neurointermediate. _ lobe and 12SI-labeled synthetic peptides. Kd The Journal of Neuroscience Rat Hypothalamic Pro-ACTH/Endorphin-Derived Peptides 841

Table 1. Molecular weight distribution of hypothalamic pro-ACTH/endorphinderived peptides

Total immuno- Apparent reactivity Radioimmunoassav Product K M, (9/o) Amino-terminalACTH Pro-ACTH/endorphin 0.09 26,000 n.d.a 0.22 16,000 3 0.41 6500 5 ACTH 0.56 4600 2 wMSH 0.87 1600 90 fl-Endorphin Pro-ACTH/endorphin 0.09 26,000 1 @-Lipotropin 0.38 8800 4 @-Endorphin 0.63 3500 95 Carboxyl-terminalACTH ACTH biosynthetic 0.14 21,000 2 Intermediate GlycosylatedCLIP 0.56 4600 20 CLIP 0.72 2500 80 y-Lipotropin P-Lipotropin 0.39 8500 4 y-Lipotropin 0.59 4100 80 0.12 2500 12 Joiningpeptide 0.39 8500 1 Joiningpeptide 0.79 1900 98 T-MSH 0.44 7100 12 Glycosylatedr,MSH 0.65 3300 83 The data presented represent a summary of the Sephadex G-75 gel filtration analyses shown in Figure 2. Data are expressed as percentage of total immunoreactivity recovered for a given radioimmunoassay. The distribution coefficient (K,,) is defined as ( V. - V,)l( V, - VJ, where V. represents the elution volume of a given molecule, and V, and V. represent the total and void volumes, respectively. 0 n.d. = Not detected.

munoreactivity (Fig. 2, middle; Table l), correspondingto the dorphin (Fig. 2, top) assays,the total amount of y-LPH-related molecular weights of CLIP [ACTH( l&39)] and glycosylated material per hypothalamus is equimolar to the total amount of CLIP, were detected. Whereasthe elution position of ACTH is fl-endorphin-related material per hypothalamus. identicalto that of glycosylatedCLIP, the relatively small amount The amino-terminal 94 is composedof amino acids of pro- of ACTH detected with the amino-terminal ACTH assay(Fig. ACTH/endorphin, a cystine-rich region at the extreme amino 2, top), demonstratesthat most of the material eluting in this terminus, a region referred to as y,-MSH in the middle, and a region representsglycosylated CLIP. The percentageof hypo- joining or “hinge” peptide region at the extreme carboxyl ter- thalamic CLIP-related immunoreactive material that corre- minus (Drouin and Goodman, 1980; Nakanishi et al., 1979). spondsto a glycosylated CLIP-sized moleculeis identical to that The carboxyl terminus of rat-joining peptide containsa potential found for CLIP synthesized in the rat intermediate pituitary amidation site (-Glu-Gly-Lys-Arg-), and amidatedjoining pep- (Bennett et al., 1982; Eipper and Mains, 1982). A minor peak tide has been identified in extracts of human pituitary (Seidah eluted in the region of ACTH biosynthetic intermediate, rep- et al., 1981). In hypothalamic extracts, one major peak, corre- resentingless than 2% of the total ACTH-related immunoreac- sponding to the size of rat-joining peptide (1883 Da), was de- tivity. The antiserum usedin this analysisdoes not cross-react tected with an immunoassayspecific for amidatedjoining pep- with pro-ACTH/endorphin or CLIP-related peptideslacking the tide (Fig. 2, bottom; Table 1); a minor component eluted with carboxyl-terminal phenylalanine residue.The total amount of a Kd of 0.39 (inset). Similar analyseswith a general joining carboxyl-terminal ACTH-related immunoreactivity was equi- peptide immunoassay(Eipper and Mains, unpublished data) molar to that found for amino-terminal ACTH and p-endor- resulted in an identical gel filtration profile; similar amounts of phin-related material (Fig. 2, top). immunoreactive material were detected with both the general Two peaksof r-LPH-related immunoreactivity, correspond- and amide-specificjoining peptide assays,suggesting that the ing to the molecularweights ofP-LPH and -/-LPH, were detected joining peptide-sizedimmunoreactive material is largely a-ami- (Fig. 2, middle; Table 1). An additional shoulder of y-LPH- dated. The total amount of joining peptide-related immuno- related immunoreactivity, with an apparent molecular weight reactivity was roughly equimolar to that of other ACTH/en- of 2500, was also detected. This suggeststhe occurrenceof fur- dorphin-derived peptides (Fig. 2, top, middle). ther limited proteolytic processingof hypothalamic r-LPH-re- Analyses with an antiserum directed againstr-MSH detected lated immunoreactive material. Although precisequantitation two peaksof r-MSH-related immunoreactivity (Fig. 2, bottom; of rat y-LPH-related immunoreactivity is not possiblebecause Table 1) correspondingto the elution positions of glycosylated of the speciesspecificity of the assay,the percentageof @-LPH- T,-MSH and of a molecule with an apparent molecular weight sized molecules,compared with total immunoreactivity, was of 7100 (K., = 0.44). In three separateanalyses, the speciesof the samewhether the y-LPH or /3-endorphinimmunoassay was Kd 0.44 constituted an average of 18% of the total r-MSH- used. If one equatesthe amount of ,&LPH-sized material per related immunoreactivity. The molecular weight differencebe- hypothalamusdetected by the y-LPH (Fig. 2, middle) and P-en- tween the speciesof Kd 0.39 detected with the joining peptide 842 Emeson and Eipper Vol. 6, No. 3, Mar. 1986

Figure 3. Gel filtration analysis of hypothalamic extracts using modification-specific LU-MSH antisera. Tissue extracts from 38 adult male rat hypothalami were fractionated on Seph- adex G-75 as described in Materials and Methods. Aliquots (SO-300 ~1) were analyzed by radioimmunoassay utilizing a general amino-terminal ACTH assay (0). an amide-directed (u-MSH assay (0), and an acetyl-directed (u-MSH assay (A). Arrows mark the elution positions ofeither )H-labeled peptides obtained from a mouse corticotropic tumor cell line (AtT-20) or lz51-labeled synthetic peptides. Recovery of general II III Illtl,,,,ll,,,,,,,,,,,,,,,,,,, amino-terminal ACTH-related immu- IO 20 30 40 50 noreactivity was 9OOVo. FRACTION NUMBER immunoassay and that of Kd 0.44 detected with the y,-MSH more recent studieshave reported the presenceof exclusively assay roughly corresponds to the molecular weight of joining desacetyl-cY-MSH[ACTH( l-l 3)NH,] in extracts of whole brain peptide. The gel filtration profiles obtained with both the y- (Evans et al., 1982) and extracts of rat hypothalamus (Dennis MSH and joining peptideimmunoassays were identical to those et al., 1983a, b, Evans et al., 1982; Geis et al., 1984; Liotta et found for rat pars intermedia (data not shown). Previous studies al., 1984; Smith et al., 1982; Turner et al., 1983).Three LU-MSH have demonstratedthat 50 to 70% of the N-terminal fragment antisera directed against different c~-MSH antigenic determi- synthesized by pituitary melanotropes is glycosylated pro-ACTH/ nants were used for initial analysis of hypothalamic extracts endorphin(l-74), the remainder being cleaved to form glyco- fractionated by gel filtration (Fig. 3). The general amino-ter- sylated Lys--r,-MSH and pro-ACTH/endorphin( 149) (Seger and minal ACTH antiserum demonstrated the expected peak of Bennett, 1984). a-MSH-sized immunoreactive material, representing8 5% of the total ACTH-related immunoreactivity (similar to Fig. 2, top). Characterization of a-MSH-sized material When the a-MSH-sized material was analyzed with an amide- Although earlier studies reported the presence of significant directed LU-MSHantiserum, similar amountsof immunoreactive amounts of authentic (u-MSH [cY-N-acetyl-ACTH( l-l 3)NH,] in material were detected. An acetyl-directed (r-MSH antiserum whole brain extracts (Loh et al., 1980; O’Donohue et al., 198 l), detected only 13% as much immunoreactive material as the

40 ACTH(I - 13)NH2 I Figure 4. Reverse-phase HPLC analysis of hypothalamic cr-MSH-sized material. A pool of cu-MSH-sized material ACTH( I - 13 lNH2 (K, 0.79-0.9 1) from extracts of 26 adult 30 sulfoxidr male rat hypothalami was analyzed on s a Waters C,, PBondapak column equil- 5 > 25 ibrated with 4% acetonitrile in 0.1% tri- I- fluoroacetic acid. Peptides were eluted 5 with a linear gradient from 4 to 24% c acetonitrile in 0.1% trifluoroacetic acid :: 20- over 10 min, followed by a linear gradient from 24 to 48% acetonitrile in i 0.1% trifluoroacetic acid over 50 min. 20 15- For immunoassays, aliquots (200 pl) of ; 1 ml fractions were analyzed with a gen- z eral amino-terminal ACTH assay f#), - IO- au amide-directed (r-MSH assay(O), and diocrtyl- CYMSH an acetyl-directed LU-MSH assay (A). Ar- rows mark the elution positions of syn- 5- thetic o-MSH-related peptides, which were determined in a parallel RP-HPLC analysis by monitoring absorbance at o- 220 nm. Recovery of general amino- terminal ACTH immunoreactivity was 30 35 40 45 50 55 60 94%. FRACTION NUMBER The Journal of Neuroscience Rat Hypothalamic Pro-ACTH/Endorphin-Derived Peptides 843

B--endo(l-31)

Fraction number Figure 5. Analysis of ,3-endorphin-sized material on SP-Sephadex. A pool of /3-endorphin-sized material from extracts of 38 adult male rat hvoothalami-a r -~~- \(IL Y 0.54477) was dried and resuspended in 50% acetic acid: ‘H-Tvr-labeled AtT-20 B-endorphiml-31) was added (co.05 nmol) and the sample was applied to a SP-Sephadex column (0.75 x 20.5 cm). One millihter fractions were‘collected (flow rate 9-10 ml/hrj. The eiution position of the ‘H-labeled AtT-20 fi-endorphin(1-31) was determined by liquid scintillationcounting of 100 pl aliquotsof eachfraction. For immunoassay, aliquots (30 ~1) were pooled by fives (fractions l-30 and fractions 90-100) or pairwise (fractions 3 l-89). Recoveries of radioactivity and immunoreactivitywere 72 and 77%,respectively. Arrows mark the elution positionsof P-endorphin(l-26)(220 mM NaCl),@-endorphin(l- 27) (270 mM NaCl), and 6-endorphin(13 1) (360 mM NaCl). The elution positions of AC-B-endorphin(l-26), Ac-&endorphin(l-27), and AC-@- endorphin(l-3 1) correspondto fractions3 1, 39, and 6 1, respectively.fi-Endoxphin( l-3 l), @-endorphin(l-27), and &endorphin(l-26) represented 56, 30, and 1l%, respectively,of the total hypothalamic@-endorphin-related immunoreactive material. other two antisera. These results suggestthat hypothalamic of the p-endorphin-sized immunoreactive material from ex- a-MSH-sized material is fully a-amidated at its carboxyl ter- tracts of normal rat brain was cy-N-acetylated.The P-endorphin- minal, whereasonly a small percentageof the moleculesare LY- sizedmaterial from rat hypothalamuswas characterized by ion- N-acetylated. exchange chromatography on a column of SP-Sephadex(Fig. In order to more fully characterize the cr-MSH-sized material 5). A trace amount of 3H-labeled&endorphin( l-3 1) obtained from rat hypothalamus, the cu-MSH-sized peptides were ana- from a mouse pituitary corticotropic tumor cell line (AtT-20) lyzed by RP-HPLC in the trifluoroacetic acid/acetonitrile sys- was included in each sample to mark the elution position of tem of Bennett et al. (1981b), which separatescu-MSH-related &endorphin( l-3 1). We detected three peaksof&endorphin im- moleculesprimarily by their state of acetylation (Fig. 4). The munoreactivity with elution positions correspondingto those major immunoreactivecu-MSH-related peptides were elutedwith of &endorphin( l-3 l), &endorphin( l-27), and @-endorphin(l- the retention times of ACTH(l-13)NH, or its sulfoxide and 26) or acetyl-@-endorphin(l-27) (Fig. 5); the latter two peptides represented85% of the total a-MSH-related immunoreactivity. are not resolved by this system. Owing to the absenceof sig- Smaller peaks eluted at the positions of (r-MSH [cr-N-acetyl- nificant amounts of the cy-N-acetylatedderivatives of /3-endor- ACTH( l-l 3)NH,] and its sulfoxide representedapproximately phin( l-3 1) and &endorphin( l-26), the earliest-elutedform of 13%of the Lu-MSH-relatedimmunoreactive material. Radioim- /3-endorphin-relatedmaterial was tentatively identified as@-en- munoassaysof the samefractions with the amide-specific(u-MSH dorphin( l-26), rather than acetyl-D-endorphin(l-27). antiserum gave identical results, and assayswith the acetyl- In the analysisshown, the cu-N-acetylatedforms of @-endor- specific(u-MSH antiserum further confirmed the identification. phin-primarily acetyl-P-endorphin( l-3 1)-represented less In seven separateanalyses of a-MSH-sized material by RP- than 2% of the total P-endorphin-related immunoreactivity. In HPLC, the amount of immunoreactive material corresponding four separateanalyses, the amount of @-endorphin-relatedim- to (Y-MSH or its sulfoxide varied between 3 and 13%of the total munoreactive material corresponding to the expected elution cu-MSH-relatedimmunoreactivity. The occurrence of substan- position of cr-N-acetyl-&endorphin( l-3 1) varied between 1 and tial amountsof the sulfoxides of both ACTH( l-l 3)NH, and (Y- 6%. Full-length P-endorphin(l-3 1) (acetylated and not) consis- MSH in hypothalamic extracts, rather than representing the tently acounted for about 60% of the p-endorphin-sized im- actual physical form of thesemolecules as they exist in hypo- munoreactive material, with the remaining 40% accounted for thalamic tissue,probably resultsfrom oxidation during the course by material that had undergonecarboxyl-terminal proteolytic of biochemical manipulation. processing. The extent of carboxyl-terminal proteolysis was somewhat more variable, the ratio of /3-endorphin(l-27) to Characterization of (?-endorphin-sizedmaterial @endorphin(l-26) varying between 1 and 2. Zakarian and Smyth (1982a, b) have reported region-specific Although ion-exchangechromatography utilizing SP-Sepha- processingvariations of /I-endorphin throughout the CNS, re- dex representsan effective means for separating various a-N- vealing two contrasting patterns of a-N-acetylation, which sug- acetylated and carboxyl-terminal shortenedforms of P-endor- gestthe gradual increaseof suchacetylation from hypothalamus phin, the presence of relatively high concentrations of NaCl in to midbrain. Weber et al. (1981) have found that lessthan 2% the column fractions may interfere with subsequentimmu- 844 Emeson and Eipper Vol. 6, No. 3, Mar. 1986

B --endo( l-

,?I---endo(l-27)

P--endo(l-26)

0 10 20 30 40 50 60 70 60 so 100 Fraction number Figure 6. Analysis of P-endorphin-sized material by ion-exchange (IEX)-HPLC. A pool of@-endorphin-sized material (Kd 0.58-0.76) from extracts of 17 adult malerat hypothalamiwas dried underreduced pressure and resuspendedin 10 mM ammoniumformate, pH 2.5, in 30%acetonitrile; a 3H-labeled sample of AtT-20 p-endorphin(l-31) was added as an internal standard (10.05 pmol). The sample was analyzed by IEX-HPLC as described in Materials and Methods. For immunoassays, aliquots (100 ~1) of 0.5 ml fractions were pooled by fives (fractions l-25 and 86-95) or pairwise (fractions 26-85), dried under reduced pressure, and dissolved in 100 mM sodium phosphate, pH 7.6, containing 1% Triton X-100. The elution position of the ‘H-labeled marker was determined by liquid scintillation counting of 50 pl aliquots of each fraction. Recovery of radioactivity was 73%. Arrows mark the elution positions of ,&endorphin(l-26), fl-endorphin( l-27), and p-endorphin( l-3 1). The elution positions of acetyl-fi- endorphin( l-26), acetyl-fi-endorphin( l-27), and acetyl+-endorphin( l-3 1) correspond to fractions 37,42, and 68, respectively. fl-Endorphin( l-3 l), acetyl-fl-endorphin(l-31), &endorphin(l-27), and fl-endorphin(l-26) represented 57, 7, 19, and 14%, respectively, of the total hypothalamic fl- endorphin-related immunoreactive material.

noassay analyses.This problem is particularly evident when lated rat CLIP at pH 4.47, were detected; the theoretical iso- attempting to analyze a tissue such as hypothalamus, whose electric focusing points for the phosphorylated and nonphos- fl-endorphin content is low when compared to the pituitary. phorylated forms of rat CLIP are 4.08 and 4.43, respectively Separation of the various forms of /3-endorphinby IEX-HPLC (Eipper and Mains, 1982). The minor peaksof immunoreactiv- with volatile buffers was investigated as an alternative fraction- ity focusing at pH 3.68 and pH 3.05, which did not correspond ation method (Fig. 1). Whole hypothalamic extracts or ,&en- to any tritium counts from the pituitary standard, may be due dorphin-sized material were analyzed by ion-exchange HPLC to the presenceof glycosylated CLIP in the original G-75 gel on a cation-exchange column; peptides were eluted from the filtration pool. Basedon the distribution of immunoreactivity column with an increasing concentration of ammonium for- in Figure 7 (top), >90% of the hypothalamic CLIP-sized ma- mate, pH 2.5, in 30% acetonitrile (Fig. 6). Four major peaksof terial is phosphorylated. In four separateanalyses, the extent of p-endorphin-related immunoreactivity, corresponding to the phosphorylation of hypothalamic CLIP-sized material varied elution positionsof p-endorphin( l-3 l), acetyl-p-endorphin( l- between 83 and 93%. 3 l), fi-endorphin( l-27), and &endorphin( l-26), were detected Alkaline phosphatasetreatment was used to verify the hy- (Fig. 6). Minor peaks correspondedto the elution positions of pothesisthat the major difference between the two isoelectric acetyl-&endorphin( l-26) (fraction 37) and acetyl-fi-endor- forms of CLIP was the presenceof a phosphateresidue (Fig. 7, phin( l-27) (fraction 42). Resultsobtained using IEX-HPLC were bottom). When hypothalamic CLIP-sized material was treated consistent with those previously obtained with SP-Sephadex, with alkaline phosphatasebefore application to the isoelectric demonstrating that fully bioactive p-endorphin( l-3 1) accounts focusing gel, a single major peak of CLIP-related immunoreac- for more than half of the total hypothalamic P-endorphin-re- tivity, focused at pH 4.47, was obtained; alkaline phosphatase lated immunoreactivity. treatment also converted the radiolabeled pituitary CLIP internal standardinto a singlepeak, focusing at pH 4.47. The quan- Characterization of CLIP-sized material titative conversion of the more acidic form of hypothalamic Previous studies have demonstrated that approximately two- CLIP to the more basic form by treatment with alkaline phos- thirds of the CLIP-sized material in the rat intermediate pitu- phatasedemonstrates that the major difference between these itary is phosphorylatedat Ser31(Bennett et al., 1982; Browne et isoelectricforms is the presenceof a phosphatemoiety. al., 1981 a; Eipper and Mains, 1982). In order to determine the In order to confirm the extent of phosphorylation of CLIP extent of phosphorylation of the CLIP-related moleculesin rat in hypothalamic tissue extracts, tryptic digestsof control and hypothalamus, CLIP-sized material was fractionated by iso- alkaline phosphatase-treated CLIP-sized material were analyzed electric focusing (Fig. 7, upper). A trace amount of jH-labeled as in Figure 7 (data not shown). Approximately 82% of the CLIP obtained from primary cultures of rat neurointermediate CLIP-related immunoreactivity focusedat pH 3.12, closeto the lobe was included in each sampleas an internal standard. Two theoretical isoelectric point for phosphorylated ACTH(22-39) peaks of CLIP-related immunoreactivity, which focused with (PI = 3.18), with the remainder focusing at pH 3.70, which is phosphorylatedrat CLIP at pH 4.12 and with nonphosphory- near the theoretical isoelectric point for nonphosphorylated The Journal of Neuroscience Rat Hypothalamic Pro-ACTH/Endorphin-Derived Peptides 845

6. ,5,5

5.0

,380

2,5

Figure 7. Isoelectric focusing of hypothalamic CLIP-sized immunoreactive material. A pool of CLIP-sized material (& 0.69-0.79) from extracts of 48 adult rat hypothalami was dried, resuspended in 0.1 M NH,HCO, containing 0.5 mg/ml BSA; a sample of 3H-phenylalanine-labeled CLIP (10.08 pmol), obtained from primary cultures of rat intermediate pituitary, was added. The sample was then di- vided into equal aliquots; one aliquot was analyzed directly (top) and the other was digested with alkaline phosphatase before analysis (bottom). The samples were applied to a 2 mm layer of 20 Sephadex IEF resin (pH 2.5-5.0) in the region of slice 25. Samples were eluted from the resin in 2 ml of 25 mM NH,HCO,, 0.1 mg/ml BSA, and 0.005% NP-40. The isoelectric focusing point of the ‘H-labeled intermediate pituitary 60 CLIP was determined by liquid scintillation counting of 700 pl aliquots of each fraction. For immunoassays, samples were dried under reduced pressure and dissolved in 100 mM sodium phosphate, pH 7.6, containing 1% Triton X- 3 100. Recoveries of immunoreactivity for the control and alkaline phospha- 0 5 IO I5 20 25 30 tase-treated sample were 99% and 84%, Slice number respectively.

ACTH(22-39) (PI = 3.52). Alkaline phosphatasetreatment of modified -y-LPH to approximately 4.17. The majority of the the carboxyl-terminal tryptic peptides resultedin a singlemajor hypothalamic y-LPH-sized material is indistinguishablefrom peak, focusingat pH 3.70. The resultsobtained by analysis of -y-LPH isolated from the pars intermedia, and doesnot appear intact CLIP or the carboxyl-terminal tryptic peptide of CLIP to be subject to additional posttranslational processing. were in agreement. Discussion Characterization of y-LPH-sized material Publishedreports on the ACTH and P-LPH regionsof the pro- Hypothalamic y-LPH-sized immunoreactive material was frac- ACTH/endorphin precursor indicate that the proteolytic pro- tionated by isoelectric focusing in order to determine whether cessingpattern in the hypothalamus closely resemblesthat in it is subjectedto additional posttranslationalmodifications, such the intermediate pituitary (Bameaet al., 1981, 1982; Gramsch asacetylation, phosphorylation, or sulfation, which would affect et al., 1980; Lob et al., 1980; 01~011et al., 1978; Seizingeret its isoelectric point (Fig. 8). A single major peak, representing al., 1984). In the present studies,this similarity was extended 74% of the total y-LPH-related immunoreactivity, focusedat to the amino-terminal region of pro-ACTH/endorphin; at the pH 4.32, the theoretical isoelectricfocusing point for rat y-LPH level of gel filtration, material of the size of joining peptide and (Eipper and Mains, 1982). A minor peak focusing at pH 4.44, glycosylated y,-MSH representedmajor end products. The ma- representingapproximately 10% of the total y-LPH-related im- jor peptide products found in the rat hypothalamus are dia- munoreactivity, could result from the removal of either the grammed in Figure 9; the cystine-rich amino-terminal region carboxyl-terminal asparticacid or the amino-terminal glutamic was not investigated in thesestudies and is therefore not rep- acid residues.Addition of an cu-N-acetyl, phosphate, or sulfate resented. The endoproteolytic processingevents that proceed moiety would be expected to shift the isoelectric point of the essentially to completion all involve cleavage at a -Lys-Arg- 848 Emeson and Eipper Vol. 6, No. 3, Mar. 1986

Figure 8. Isoelectric focusing of hy- :: 1.0 2,5 pothalamic y-LPH-sized immunoreac- z tive material. A pool of r-LPH-sized material (K,, 0.52-0.62) from extracts P of 18 adult male rat hypothalami was 1 dried under reduced pressure, resus- -f pended in 5 mM acetic acid, and applied .I 5 to a 1 mm layer of Sephadex IEF (pH 2.5-5); samples were applied in the region of slice 25. Electrophoresis was allowed to proceed for 19 hr at 600 V and the bands of resin were eluted in 3 ml of 50 mM NH,HCO, containing 1 mp/ ml BSA and 0.1% NP-40. For immunoassays, samples were treated as de- I I I I I I I, 1 , , , , , ,, , , , ,, , , , , , scribed in Figure 7; recovery of -/-LPH- 0 IO 20 30 related immunoreactivity was 120%. SLICE NUMBER sequence,except for the cleavagethat generatesthe amino ter- Previous studieshave not reached a consensusregarding the minus of joining peptide, which involved cleavage at an -Arg- extent to which a-MSH- and /3-endorphin-relatedmaterial in Arg- site. The proteolytic processingat the -Arg-Lys- sequence extracts of rat hypothalamus or whole brain is a-N-acetylated separatingthe cystine-rich amino-terminal region from y,-MSH (Loh et al., 1980; O’Donohue et al., 1982). The biochemical proceededonly partially, as in the pars intermedia (Segerand analysespresented here demonstrate that about 10%of both the Bennett, 1984). hypothalamic LU-MSH-and p-endorphin-related material rep- The cu-N-acetylationof ACTH( l-l 3)NH, resultsin a 15fold resentsa-N-acetylated material (Figs. 3-6). These resultsare in increasein melanophorestimulating activity, enhancespotency agreementwith a majority of the previously published works in behavioral tests, such as excessivegrooming and visual dis- regarding the limited extent to which both CY-MSHand ,&en- crimination, and decreasesability to block opiate-induced an- dorphin are acetylated in the hypothalamus (Evans et al., 1982; algesia(Akil et al., 1980; Guttmann and Boissonnas, 1961; Geis et al., 1984; Liotta et al., 1984; Smith et al., 1982; Turner O’Donohue et al., 198 1, 1982). The amino-terminal acetylation et al., 1983; Weber et al., 1981; Zakarian and Smyth, 1979, of P-endorphin destroys its opiate activity (Akil et al., 1981; 1982a,b). If, ashas been suggested for the intermediate pituitary Smyth et al., 1979). The presenceor absenceof a posttransla- (Chappell et al., 1982; Glembotski, 1982), a single secretory tional modification such as a-N-acetylation thus provides a granule-associatedacetyltransferase is responsiblefor the ami- mechanismfor modulatingthe hormonal or neurosecretoryout- no-terminal acetylation of both (u-MSH and /3-endorphin, par- put of neuronsexpressing the samegene. allel alteration in the acetylation of both products would be

- Gly - Lyr-Lyr-Arg-Arg-

-Gly-Lyr-Arg- -Arg-Lys- -Arg-Arg- \ / -Lyr-Arg- -Lyr-Arg- -Lyr-Lyr- / \/ \/ \/ \

Figure 9. Major products of pro-ACTH/endorphin processing in rat hypothalamus. A schematic diagram of the rat pro-ACTH/endorphin molecule is depicted (Drouin et al., 1980). It shows the major end products of pro-ACTH/endorphin processing in rat hypothalamus. The amino acid sequences at potential paired basic amino acid cleavage sites and at potential amidation sites are designated. The N-linked oligosaccharide groups within y,-MSH and CLIP, the phosphate moiety within CLIP, and the site of cleavage producing the amino terminus of T,-MSH (Lys,,-ra-MSH) have been positioned by analogy to the pituitary (Bennett et al., 1981a; Browne et al., 1981b; Eipper and Mains, 1977). The Journal of Neuroscience Rat Hypothalamic Pro-ACTH/Endorphin-Derived Peptides a47 expected. Approximately 80% of the cu-MSH- and 60% of the not cross-reactwith ACTH( 18-38)-relatedmolecules, this result @-endorphin-relatedmaterial in extracts of rat or guinea pig suggeststhat carboxyl-terminally shortenedCLIP doesnot rep- caudal medulla, containing the region of the nucleusof the sol- resenta major hypothalamic product. itary tract, is a-N-acetylated (Dores et al., in press).Liotta et As summarizedin Figure 9, the differencesin the processing al. (1984), using in vivo labeling, reported production of only patterns in the intermediate pituitary and the hypothalamus nonacetylated forms of both CY-MSHand @-endorphinby neu- might be explained by differencesin only three enzymatic ac- ronsin the region of the arcuatenucleus. These two observations tivities involved in posttranslationalprocessing. Hypothalamic suggestthat the cu-N-acetyl-ACTH( l-l 3)NH, and cu-N-acety- ACTH/endorphin neurons, similar to intermediate pituitary lated P-endorphin in the hypothalamic region may be present melanotropesat the level of grossproteolytic processing,appear in nerve fibersor terminal fields originating in the nucleustrac- more like anterior pituitary corticotropesin exhibiting relatively tus solitarius. Alternatively, a subpopulation of ACTH/endor- little a-N-acetylation of wMSH and P-endorphin. Carboxyl- phin arcuate neurons projecting to specific target areas may terminal proteolysis of p-endorphin, which does not occur to contain peptide products different from thoseelaborated by the any significant extent in corticotropes, occursto a lesserextent majority. in hypothalamic neuronsthan in melanotropes.In addition, the Limited carboxyl-terminal proteolysis of p-endorphin(l-3 1) hypothalamic neuronsexhibit more extensive phosphorylation has pronouncedeffects on opiate activity (Geisow et al., 1977; of CLIP than is observedin either melanotropesor corticotropes. Hammondset al., 1984;Smyth et al., 1979).In opiate bioassays, The fact that nearly equimolar amounts of /3-endorphin-, @endorphin(l-27) has been shown to be .approximately equi- T-LPH-, a-MSH-, CLIP-, and joining peptide-related immu- potent to @-endorphin(l-31) in inhibiting the contraction of noreactivity are found in the hypothalamus, and that suchmol- guineapig myenteric plexus(M&night et al., 1983).In the CNS, eculeshave been localized to the samesecretory granules (Bar- the analgesicpotency of P-endorphin(l-27) is only 2% that of neaet al., 1981; Pelletier, 1979),indicates that significantamounts p-endorphin(l-3 l), but this decreasedopiate bioactivity is only of eachof the various product peptidesare secretedtogether in partially reflectedin a decreasedaffinity for opiate receptorsand responseto appropriate stimuli, and that the coordinate effects makesp-endorphin( l-27) a potent antagonistofp-endorphin( l- of the various peptideson the sameand different target neurons 3 1) (Hammondset al., 1984). must be considered. The results presentedhere are in agreementwith previous studies demonstrating that p-endorphin( l-3 1) representsthe References predominant end product in the hypothalamus (Dennis et al., Akil, H., W. A. Hewlett, J. D. Barchas, and C. H. Li (1980) Binding 1983a;Liotta et al., 1984;Zakarian and Smyth, 1979, 1982a,b). of ‘H-beta-endorphin to rat brain membranes: Characterization of Ion exchangechromatography (Figs. 5 and 6) hasdemonstrated opiateproperties and interaction with ACTH. Eur. J. Pharmacol. 64: that, in addition to @endorphin(l-3 l), p-endorphin( l-27) and 1-8. &endorphin( l-26) represent significant end products. The re- Akil, H., Y. Ueda, H. L. Lin, and S. J. Watson (1981) A sensitive sultsof in vivo biosynthetic labelingstudies suggested that only coupled HPLC/RIA technique for the separation of endorphins: Mul- intact &endorphin( l-3 1)was a major product peptide of arcuate tiple forms of @-endorphin in rat pituitary intermediate vs. anterior ACTH/endorphin neurons (Liotta et al., 1984); however, as- lobe. Neuropeptides I: 429-446. suming that the kinetics of carboxyl-terminal shortening are Bamea, A., G. Cho, and J. C. Porter (1981) Apparent coquestration of immunoreactive corticotropin, cu-melanotropin and y-lipotropin comparablein hypothalamus and pars intermedia, the labeling in hypothalamic granules. J. Neurochem. 36: 1083-1092. period studied by Liotta et al. (1984) would not have been Bamea, A., G. Cho, and J. C. Porter (1982) Molecular-weight profiles sufficientto determinethe final extent of carboxyl-terminal pro- of immunoreactive corticotropin in the hypothalamus of the aging teolytic processingof p-endorphin (Glembotski, 1982).As with rat. Brain Res. 232: 355-363. cu-N-acetylationof P-endorphin, the degreeto which the car- Bennett, H. P. J., A. M. Hudson, L. Kelly, C. McMartin, and G. E. boxyl-terminally shortenedforms of /3-endorphinare produced Purdon (1978) A rapid method, usingoctadecasilyl-silica, for the may representone of the regulatory mechanismsby which nor- extraction of certain peptides_ _ from tissues. Biochem. J. 175: 1139- mal levels of opiate activity are maintained within the CNS. 1141. The phosphorylationof corticotropin-related peptideshas been Bennett, H. P. J., C. A. Browne, and S. Solomon (198 la) Biosynthesis of phosphorvlated forms of corticotronin-related _Dentides. - Proc. Natl. shown to vary widely among speciesand also among tissues Acad. Sci. USA 78: 4713-4717. - within a given species;one-fifth of the mousepituitary ACTH Bennett, H. P. J., C. A. Browne, and S. Solomon (1981 b) Complete and CLIP, one-third of the human pituitary ACTH, one-half of purification of pituitary peptides using reversed-phase HPLC alone. rat anterior pituitary ACTH, and two-thirds of rat intermediate In Peptides,D. H. Rich and E. Gross, eds., pp. 785-788, Pierce pituitary CLIP are phosphorylated(Bennett et al., 1982;Eipper Chemical Co., Rockford, IL. and Mains, 1982;Mains and Eipper, 1983).Isoelectric focusing Bennett, H. P. J., C. A. Browne, and S. Solomon (1982) Character- analyseshave demonstratedthat 83 to 93% of rat hypothalamic ization of eight forms of corticotropin-like intermediary lobe peptide CLIP-sized immunoreactive material is phosphorylated (Fig. from the rat intermediary pituitary. J. Biol. Chem. 257: 10,096- 10,102. 7). Glycosylation of corticotropin-related peptideshas also been Bennett, H. P., C. A. Browne, and S. Solomon (1983) Alpha-N-acetyl- shownto be species-specific;whereas approximately two-thirds &endorphin( l-26) from the neurointermediary lobe of the rat pitu- of the corticotropin-related peptidesin the mousepituitary are itary: Isolation, purification and characterization by high-perlor- glycosylated,only one-sixth are glycosylated in the rat (Bennett mance liauid chromatography. Anal. Biochem. 128: 12 l-l 29. et al., 1982; Eipper and Mains, 1982). The present study dem- Bloom, F. E., E. L. F. Bat&berg, T. Shibasaki, R. Benoit, N. Ling, and onstratesthat approximately 20% of the hypothalamic carboxyl- R. Guillemin (1980) Localization of y-melanocyte-stimulating hor- terminal ACTH-related immunoreactivity correspondsto the mone (-/-MSH) immunoreactivity in rat brain and pituitary. Reg. molecularweight ofglycosylated CLIP (Fig. 2, middle), the same Peptides 1: 205-222. percentagefound in rat intermediate pituitary (Bennett et al., Browne, C. A., H. P. J. Bennett, and S. Solomon (1981a) Isolation 1982;Eipper and Mains, 1982).The physiological consequences and characterization of corticotropin- and melanotropin-related peptides from neurointermediate lobe of rat pituitary by reversed-phase of suchlimited glycosylation and extensive phosphorylation re- liquid chromatography. Biochemistry 20: 4530-4537. main to be determined. Equimolar amounts of CLIP-related Browne, C. A., H. P. J. Bennett, and S. Solomon (198 1b) The isolation moleculesand other pro-ACTH/endorphin-derived peptides and characterization of y,-melanotropin from the neurointetmediary were found in the hypothalamus (Fig. 2, middle). Although the lobe ofthe rat pituitary. Biochem. Biophys. Res. Commun. 100: 336- carboxyl-terminal ACTH antiserumused in theseanalyses does 343. 848 Emeson and Eipper Vol. 6, No. 3, Mar. 1986

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