Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3845-3849, May 1993 Neurobiology Purification of a receptor from pig brain YAOHUI CHEN*, ALAIN FOURNIERt, ALAIN COUVINEAU*, MARC LABURTHE*, AND BRIGITTE AMIRANOFF*t *Laboratoire de Biologie and Physiologie des Cellules Digestives, Institut National de la Sant6 et de la Recherche Mddicale, U 239, 16 Rue Henri Huchard-75018 Paris, France; and tUniversitd du Quebec, Institut National de la Recherche Scientifique, INRS-Santd, 245 Boulevard Hymus, Pointe Claire, Qudbec, H9R1G6, Canada Communicated by Tomas Hokfelt, January 4, 1993

ABSTRACT A protein was solubilized lished data), we report the purification of a galanin receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane- from pig brain, a rich source of receptors that is available in sulfonate (CHAPS) from pig brain membranes and then pu- large amounts. This represents a basic step toward knowl- rified by single-step affinity chromatography. The product edge of the pharmacology and biochemistry of galanin re- exhibits saturable and specific binding for galanin with a ceptors and should lead to a better understanding of their binding activity of 17 nmol/mg of protein and a dissociation expression in the organism. constant (Kd) of 10 nM. This represents a 300,000-fold puri- fication over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding METHODS activity. Gel electrophoresis of the affinity-purified material Materials. Synthetic porcine galanin, , vasoactive showed a single polypeptide of 54 kDa by silver staining and intestinal , synthetic , , baci- after radioiodination. Cross-linking of a purified fraction af- tracin, , pepstatin A, GTP, GDP, guanosine 5'-[13,v- rmity-labeled with 125I-labeled galanin revealed a single band imido]triphosphate, cholesteryl hemisuccinate, 3-[(3-cholami- for the galanin-receptor complex at 57 kDa. The general dopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), binding characteristics ofthe purified preparation appeared to and phenylmethylsulfonyl fluoride were obtained from Sigma; be identical to those of the crude soluble material as far as porcine was from Novo Research Institute (Copenha- specificity toward galanin and the structural requirement for gen); SDS/PAGE chemicals and the gel silver-staining kit galanin are concerned. In contrast, unlike the CHAPS-soluble were from Bio-Rad; marker proteins were from BRL; disuc- galanin receptor, binding of 125I-labeled galanin to the purified cinimidyl tartarate (DST) was from Pierce; tert-butoxycar- galanin receptor was not sensitive to guanine nucleotides, bonyl(Boc)aminoacids andbenzotriazolyl-N-oxy-tris(dimeth- suggesting that dissociation ofthe inhibitory guanine nucleotide ylamino)phosphonium hexafluorophosphate (BOP) were from binding protein from the galanin receptor occurred during Richelieu Biotechnologies (St-Hyacinthe, Quebec, Canada); purification. The purification to homogeneity of a galanin fluoren-9-ylmethoxycarbonyl (Fmoc)-protected amino acids receptor paves the way toward its sequencing and cloning. were from Advanced ChemTech, and diisopropylethylamine (DIEA) was from Pfaltz & Bauer and was distilled from Galanin is an ubiquitous identified in porcine ninhydrin before use. Reagents used as scavengers during intestine on the basis of its amidated C terminus (1). In deprotection of the side chains (thioanisole, agreement with its widespread localization in the central and ethanedithiol, or anisole) were obtained from Aldrich. PepSyn peripheral nervous system (2), galanin elicits a wide range of Gel, a polyamide-based support functionalized with sarcosine biological responses (3). Since the discovery of galanin as a ester residues was from neuromodulator in the (3, 4) and a methyl (0.3 mmol/g), purchased sympathetic neuromodulator in the endocrine (5), MilliGen (Mississauga, Ontario, Canada). Synthetic porcine its mechanism of action in these organs has been the focus of galanin was radioiodinated with 125I (Amersham) by the chlo- intensive study. Thus, radiolabeled galanin has been used to ramine-T method (26), at a specific activity of700 Ci/mmol (1 identify specific galanin binding sites mostly in the endocrine Ci = 37 GBq). Under those conditions, the tracer was prob- pancreas (6-10) and brain (11, 12). In all these studies, the ably iodinated at the two tyrosine residues present in the galanin receptor was shown to be coupled to a guanine peptide. The porcine galanin fragments galanin-(2-29), gala- nucleotide binding regulatory (G) protein to initiate the nin-(3-29), and galanin-(1-15) were kindly provided by N. cascade of cellular responses through inhibition of adenylate Yanaihara (Shizuoka, Japan). cyclase (7, 13), activation of ATP-sensitive K+ channels (14, Solubilization of Galanin Receptors. Membranes from one 15), or inhibition of calcium current (16). pig brain were prepared and solubilized essentially as de- The molecular characterization of galanin receptors from scribed for rat brain (11, 17). Briefly, for solubilizing the brain and pancreas by covalent labeling with 125I-labeled galanin receptors, 30 ml of pig brain membrane suspension galanin demonstrated that the galanin receptor is a mono- (7.5 mg/ml) was incubated in 20 mM Hepes buffer (pH 7.5) meric glycoprotein of 53-54 kDa (6, 8, 10, 11). Meanwhile, containing 30 mM CHAPS, 1.8 mM cholesteryl hemisucci- the functional association of the galanin receptor with an nate, 0.1 mM phenylmethylsulfonyl fluoride, 0.01% sodium inhibitory G (Gi) protein was demonstrated (7, 9, 10, 12, 15). azide, 30% (vol/vol) glycerol, and 25 mM KCl for 30 min at More recently, the solubilization of a rat brain galanin 0°C. The suspension was then diluted in 2 vol of the same receptor in an active form, a preliminary step toward its buffer but without CHAPS and centrifuged for 60 min at purification, confirmed the physical association ofthe galanin receptor in solution with the a subunit of a Gi protein (17). Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol- In the present study, with the use of a one-step affinity 1-propanesulfonate; DST, disuccinimidyl tartarate; G protein, gua- chromatography (ref. 18 and A.F., A.C., and M.L., unpub- nine nucleotide binding regulatory protein; Gi protein, inhibitory G protein; BOP, benzotriazolyl-N-oxy-tris(dimethylamino)phospho- nium hexafluorophosphate; Boc, tert-butoxycarbonyl; DIEA, diiso- The publication costs of this article were defrayed in part by page charge propylethylamine; DMF, dimethyl formamide; Fmoc, fluoren-9- payment. This article must therefore be hereby marked "advertisement" ylmethoxycarbonyl. in accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 3845 Downloaded by guest on September 30, 2021 3846 Neurobiology: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 100,000 x g (4°C). The supernatant was used as starting tained 49 mg ofgalanin) was pretreated with 40 ml of distilled material for further purification of galanin receptors. water for 48 h, packed into a plastic column (1 x 10 cm), and Synthesis of the Galanin Matrix. The galanin matrix was equilibrated with 20 ml of 20 mM Hepes buffer (pH 7.5) obtained by synthesizing the peptide on functionalized poly- containing 10% (vol/vol) glycerol, 1 mM CHAPS, 0.12 mM acrylamide resin. A similar procedure was used to synthesize cholesteryl hemisuccinate, 1 mM EDTA, 25 mM KCI, 20 mM a hydrophobic affinity matrix for the purification of the MgCl2, 0.01% sodium azide, and a protease inhibitor mixture vasoactive intestinal peptide receptor (A.F. et al., unpub- [1 mM phenylmethylsulfonyl fluoride/leupeptin (10 mg/ lished data). liter)/pepstatin A (10 mg/liter)] (buffer A). Soluble material Resin preparation. The PepSyn Gel resin, formed by (30 ml) containing active galanin receptor (-3.8 mg ofprotein copolymerization of dimethylacrylamide with a cross-linking per ml) was loaded on the affinity column at a flow rate of 12 monomer, was functionalized with sarcosine methyl ester ml/h at 4°C and recycled overnight through the column. At (0.3 mmol/g). Prior to peptide synthesis, the support (2.4 the end of the recycling phase, the pass-through fraction, mmol, 8 g) was treated overnight with excess ethylenedi- which contained no binding activity, was collected and stored amine (250-300 ml), which provided primary amine sites as at -80°C. The resin was then washed with 100 ml of buffer attachment points. After the incorporation of ethylenedi- A until the eluent was protein-free. The galanin receptors amine into the resin, Boc-E-aminocaproic acid (Boc-Aca), a bound to the affinity gel were eluted with 36 ml of 10 mM 6-carbon spacer arm, was coupled to the support by using magnesium acetate, pH 4.0/10% glycerol/0.01% sodium dimethyl formamide (DMF) as solvent. A 3-fold excess of azide and the protease inhibitor mixture at a flow rate of 30 Boc-Aca (7.2 mmol, 1.67 g) was used for the coupling and ml/h. The fractions containing galanin receptors were im- activation of the carboxylic function was achieved with BOP mediately neutralized with 2 M Hepes and used for ligand reagent (7.2 mmol, 3.18 g) in presence of DIEA (5-fold binding or stored at -80°C. After each purification proce- excess, 12 mmol, 1.55 g, 2.1 ml) (19). dure, the affinity column was washed successively with 5 vol Loading of the first amino acid. After equilibration of the of 1 M NaCl, 5 vol of 0.2 M acetic acid, 5 vol of 20% ethanol, gel (0.6 mmol, 2 g) in methylene chloride, the Boc protection and 10 vol of 0.04% sodium azide in H20. The affinity gel was group on aminocaproic acid was removed by 40% (vol/vol) used immediately for another purification cycle or stored at trifluoroacetic acid in CH2Cl2 (one 5-min treatment and one 4°C. It could be used for at least five successive purification 20-min treatment), followed by successive washings with procedures without any detectable modification. CH2Cl2 (two washes), isopropanol (two washes), and DMF Binding and Cross-Linking of 12II-Labeled Galanin to Mem- (two washes). The C-terminal residue alanine was then branes and Soluble and Purified Receptors. Assays of binding introduced into the modified polyacrylamide resin as a Boc- to membrane and solubilized galanin receptor were carried alanyl-4-(oxymethyl)phenylacetic acid (OMPA) derivative. out as described (11, 17). For the purified receptor, 150 ,lI of This compound was synthesized by the procedure of Mitchell fractions eluted from the affinity column and containing the et al. (20). Boc-Ala-OMPA (1.8 mmol, 0.61 g) was coupled to binding activity was incubated with 50 ,ul of the pass-through the polymer by using the concomitant neutralization- fraction, which signiflcantly increased galanin binding activ- coupling step strategy (19, 21) with BOP reagent (1.8 mmol, ity (see above), and 0.5 nM 125I-labeled galanin for 16 h at 4°C 0.79 g) and DIEA (2.7 mmol, 0.35 g, 0.47 ml). in a final volume of 250 ,ul of 20 mM Hepes buffer (pH 7.5) Assembly of the peptide chain. Before being treated with containing 2% (wt/vol) bovine serum albumin, bacitracin (1 40% trifluoroacetic acid in CH2Cl2, the Boc-Ala-resin was mg/ml), and the protease inhibitor mixture. The reaction was washed successively with DMF (two washes), isopropanol terminated by fitration through Whatman GF/C glass fiber (two washes), and CH2Cl2 (two washes). Then, Boc-Leu28 filters pretreated with 0.5% polyethylenimine in water. and Boc-Gly27 were incorporated in the peptide chain by Affinity cross-linking of 125I-labeled galanin bound to mem- following the concomitant neutralization-coupling step pro- brane or soluble receptors was performed as described (11, tocol (19, 21). After the deprotection of Gly27, the resin was 17). Cross-linking of 125I-labeled galanin to the purified re- washed with CH2Cl2 (three washes) and incubated for 2 min ceptors (0.3 ,ug of protein per ml) was performed by incu- with 5% (vol/vol) DIEA in CH2Cl2. The resin was rinsed with bating the purified galanin receptor with the tracer in 20 mM CH2Cl2 (two rinses) and DMF (two rinses), and Fmoc- Hepes buffer (pH 7.5) for 16 h at 40C in the presence or Tyr(butyl)26-COOH was coupled using four equivalents of absence of 1 AM unlabeled galanin. The 125I-labeled galanin- each coupling reactants (Fmoc-amino acid, BOP, and DIEA). receptor complexes were then cross-linked by addition of All the subsequent amino acid residues were introduced in DST at a final concentration of 1 mM for 20 min at 4°C and the peptide chain as Fmoc derivatives by using the neutral- the reaction was stopped by adding 20 ,ul of ice-cold 1 M ization-coupling step strategy with BOP reagent. Side-chain Tris HCl (pH 6.8). The proteins were then precipitated with protection of a-Fmoc-amino acids was as follows: Arg a mixture of a methanol/chloroform/water, 4:1:3 (vol/vol), [4-methoxy-2,3,6-trimethylbenzene sulfonyl (Mtr)], Asn as described by Wessel and Flugge (23). Precipitates were [triphenylmethyl (Trt)], Asp (O-butyl), His (Trt), Lys (Boc), redissolved in SDS sample buffer (pH 6.8) containing 10% Ser (butyl), Thr (butyl), and Tyr (butyl). glycerol, 3% (wt/vol) SDS, and 0.001% bromophenol blue, Removal of the side-chain protecting groups. After com- boiled for 3 min at 100°C, and analyzed by SDS/PAGE. pletion of the synthesis, the peptide support was washed with SDS/PAGE. Gel electrophoresis was performed, as de- isopropanol and methanol and dried overnight in vacuo. A scribed by Laemmli (24), using a 5% polyacrylamide stacking part of the resin (2.2 g) was transferred into a round-bottom gel and a 12% polyacrylamide slab gel as described (8). The flask. Then, 75 ml of reagent R (22) [90% (vol/vol) trifluoro- gels were stained, dried, and exposed to a Trimax type XM acetic acid/5% (wt/vol) thioanisole/3% (wt/vol) 1,2- film (3M) with a 3M Trimax intensifying screen for 1-7 days ethanedithiol/2% (vol/vol) anisole] was added and the flask at -80°C. The following proteins of known molecular mass was purged with nitrogen. The flask was shaken for 7 h by were used to calibrate the gels: myosin (200 kDa), phosphor- using a wrist-action shaker. The content of the flask was ylase b (92 kDa), bovine serum albumin (68 kDa), ovalbumin poured in a fritted-glass filter and washed successively with (43 kDa), and carbonic anhydrase (29 kDa). Silver staining of 50-100 ml of trifluoroacetic acid, isopropanol, and water. gels was performed as described by Morrissey (25). After a final washing step with isopropanol, the polymeric lodination of the Purified Receptor. The purified receptor support was dried 24 h in vacuo. eluted from the affinity column was iodinated by the chlora- Affinity Chromatography of Solubilized Galanin Receptor. mine-T method (26). Briefly, 3 ml of the fractions containing The galanin-(1-29)-polyacrylamide resin (100 mg, which con- the galanin binding activity (300 ng) was precipitated with 24 Downloaded by guest on September 30, 2021 Neurobiology: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3847

ml of methanol/chloroform/water, 4:1:3 (vol/vol). The pre- A B C cipitate was dissolved in 100 ,ul of 0.25 M sodium phosphate (pH 7.5) and incubated with 0.5 mCi of Na'25I in the presence kDa of 100 ,ug of chloramine T for 1 min at room temperature (1 200 - Ci = 37 GBq). The reaction was stopped by adding 200 ,g of sodium metabisulfite. The iodinated receptor was separated 92.5 - from free 125I on a Sephadex G-50 column, using 0.2 M acetic

acid containing 0.5% bovine serum albumin and 0.03% bac- 68 -

itracin. The radioiodinated receptor was precipitated and 57 - submitted to SDS/PAGE. 43 - ADP-Ribosylation of Fractions Eluted from the Galanin

Affinity Chromatography Column. ADP-ribosylation with 29 - pertussis toxin was carried out essentially as described (10). Briefly, 1 ml of material was incubated with preactivated pertussis toxin (100 ng/ml) in 5 mM Tris-HCl (pH 7.5) Gal - + +I + containing 1 mM ATP, 100 AM GTP, 2.5 mM MgCl2, 1 mM FIG. 1. Autoradiography of cross-linked 125I-labeled galanin to dithiothreitol, and 10 ,uM [32P]NAD. After incubation for 15 membrane, CHAPS-solubilized, and purified pig brain galanin re- min at 30°C, the proteins were precipitated with methanol/ ceptors. Membranes (lanes A), CHAPS-solubilized extracts (lanes chloroform/water, 4:1:3 (vol/vol), and redissolved in SDS B), and purified galanin receptors (lanes C) were incubated with sample buffer (pH 6.8) before analysis by SDS/PAGE. 1251-labeled galanin (0.5 nM) in the absence (-) or presence (+) of 1 Protein Determination. Protein concentration of membrane ,uM unlabeled galanin (Gal). DST (1 mM) was added to the samples that were further analyzed by SDS/PAGE. The molecular mass of was and soluble material measured using the procedure of the galanin-receptor protein complex was estimated at 57 kDa. Bradford (27) with bovine serum albumin as standard. Pro- teins in the eluted fractions were determined after SDS/ receptor complex behaved as a protein of57 kDa, the labeling PAGE by densitometric scanning of silver-stained gels using of which was specific for galanin (Fig. 1, lane B). the standard molecular weight markers (see above). Receptor Purification. The data obtained from five receptor Data Analysis. Analysis of saturation and competition of purification experiments are summarized in Table 1. The galanin binding experiments was performed by the LIGAND solubilized material (30 ml of CHAPS-solubilized extract) program (28). was allowed to bind overnight to the galanin-polyacrylamide resin. This overnight recycling completely removed the 1251_ RESULTS labeled galanin binding activity in the extract. In contrast, only 3% of the protein was adsorbed on the resin. Most of Membrane and Soluble Galanin Receptors. The character- these proteins were eluted from the column after extensive istics of galanin receptors from membranes of pig brain washing with buffer A. No galanin binding activity was found compared well with those from rat brain (11, 12, 29). (i) in this eluted fraction (Fig. 2). Magnesium acetate (pH 4) was Scatchard analysis of binding data indicated the existence of used to eluate the galanin receptor from the column in view one type of high-affinity site (Kd = 0.76 nM) with a Bm., of of the previous observation that the galanin receptor is 55 fmol/mg of protein (Table 1). (ii) The galanin fragments sensitive to pH changes and that a slightly acid buffer greatly galanin-(2-29) and galanin-(1-15) competitively inhibited reduces the affinity of the receptor for (9). The total binding of 125I-labeled galanin to membranes whereas gala- amount of protein eluted from the column in fractions con- nin-(3-29) was inactive (data not shown). (iii) The binding of taining 125I-labeled galanin binding activity was estimated galanin was inhibited by guanine nucleotides in a dose- from the trace of the A280 ofthe sample. After neutralization, dependent manner and with the following rank order of the binding of 125I-labeled galanin to the purified material potency: guanine 5'-[3,y,-imido]triphosphate > GTP > GDP. eluted from the affinity column was found to be specific and The nucleotides caused an 80% reduction in the binding of saturable (Fig. 3). Parameters obtained from the correspond- 125I-labeled galanin and the IC50 values were 20 ,uM, 0.1 mM ing Scatchard plot (Fig. 3 Inset) were Kd = 10 nM and Bmax and 0.2 mM, respectively (data not shown). (iv) Cross-linking = 3 nM. From the amount of proteins estimated by silver experiments showed that the galanin-receptor complex ofpig staining, the binding capacity ofthe putative galanin receptor brain membranes behaved as a protein of 57 kDa, the labeling was calculated to be 17 nmol/mg of protein, which corre- of which was abolished by an excess of native galanin (Fig. sponds to an enrichment of 300,000 over the crude soluble 1, lane A). receptor. This value is close to the theoretical value of 18.5 Solubilization of active galanin receptors from pig brain nmol/mg calculated on the basis of one galanin binding site was achieved using the following optimal conditions: 30 mM per 54-kDa protein (Fig. 3 and Table 1). The yield of 125I1 CHAPS and 7.5 mg of protein per ml. The solubilized galanin labeled galanin binding activity recovered from the column receptors bound galanin with a high affinity (Kd = 2.78 nM) was =60% of the total activity loaded (Table 1). and a Bm,, of 56 fmol/mg of protein (Table 1). The soluble The ligand specificity of the purified galanin receptor was galanin receptor retained its sensitivity to the inhibitory investigated by analyzing the ability of various to effects of guanine nucleotides (data not shown). Cross- compete with 1251-labeled galanin binding. Binding to the linking of 125I-labeled galanin to the CHAPS-solubilized pig magnesium acetate eluate was specific to galanin, since it was brain galanin receptor showed that the soluble galanin- not displaced by structurally unrelated peptides such as the Table 1. Binding parameters of the pig brain galanin receptor Total protein, Kd, Specific activity, Fold Recovery, Preparation mg nM fmol/mg of protein purification t Membrane 225 0.76 55 1 100 Crude soluble 114 2.78 56 1 52 Purified 2.3 x 10-4 10 17 x 106 3 x 105 31 Specific activity (binding capacity) was calculated from Scatchard analysis of 125I-labeled galanin binding. Downloaded by guest on September 30, 2021 3848 Neurobiology: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) kDa 0 1 0 FIG. 4. Electrophoretic analysis . of soluble and purified receptor pro- s iOE tein. Lanes: A, crude CHAPS extract "0 X, (10 j.g of protein) was submitted di- b rectly to SDS/PAGE and the gel was la4- MM _w silver-stained; B, 300 ng of the recep- tor preparation was electrophoreti- cally resolved and visualized by silver staining; C, autoradiography of the Fraction number 29 v2.. iodinated galanin receptor after expo- sure of gel to a 3M Trimax intensify- FIG. 2. Affinity chromatography of CHAPS-solubilized galanin A B C ing screen for 12 h at -80°C. receptor. Soluble proteins (30 ml) were loaded at 4°C on the galanin- polyacrylamide resin column (1 x 10 cm) equilibrated with buffer A. revealed a single radioactive band at 54 kDa (Fig. 4, lane C), The column was washed with buffer A (fractions 1-50) and subse- confirming the homogeneity of the purified preparation. quently with 36 ml of elution buffer containing 10 mM magnesium Additional experiments were conducted, supporting the acetate (pH 4), 10%o glycerol, 0.01% sodium azide, and the protease inhibitor mixture (fractions 51-65). Chromatography was followed conclusion that the 54-kDa protein is a galanin receptor. The by automatic recording of A28o (A) and by measuring the specific neutralized magnesium acetate-eluted fractions were incu- binding of 1251-labeled galanin (0.1 nM) to aliquots of each fraction bated with 1251-labeled galanin in the absence and in the (o). Fraction volume, 2 ml; flow rate, 30 ml/h. presence ofan excess ofnative galanin. The cross-linker DST (1 mM) was added to the medium, which was analyzed by vasoactive intestinal peptide, glucagon, insulin, substance P, SDS/PAGE. Fig. 1, lane C, shows a single labeled band at 57 or neurotensin (data not shown). kDa. This band was not observed when incubation was The fractions containing 1251-labeled galanin binding activ- performed in the presence of 1 ,uM native galanin (Fig. 1, lane ity were pooled, concentrated, and analyzed by SDS/PAGE C). If one molecule of 125I-labeled galanin (3 kDa) is bound (Fig. 4, lane B). The gel showed presence of a single band of per molecule of receptor, the intrinsic molecular mass of the silver-stained material with a molecular mass of 54 kDa, purified galanin receptor is estimated at 54 kDa by affinity indicating that the protein is free of significant contaminants. labeling experiments. The 54-kDa protein was purified with the galanin affinity The relative affinities of the fragments galanin-(2-29), column, as shown by the profile of proteins present in the galanin-(3-29), and galanin-(1-15) for the purified galanin solubilized brain tissue before affinity receptor (Fig. 3), the membrane receptor (28), and the chromatography. In- CHAPS-solubilized galanin receptor in crude extracts (17) deed, Fig. 4, lane A, shows the electrophoretic pattern were similar, indicating that the structural requirement ofthe obtained with the crude CHAPS extract including a large receptor for binding its ligand was not altered by purification. number ofprotein bands with molecular masses from >100 to However, the sensitivity to guanine nucleotides of the <10 kDa. The direct radioiodination of the purified receptor purified galanin receptor was altered. Indeed, the binding of 125I-labeled galanin to the purified extract was unaffected by these compounds, indicating that the purified receptor does 100 not maintain its connection to its G protein. Attempts to identify the inhibitory a; subunit of the inhibitory G, protein in the different fractions eluted from the affinity column by '0 [32P]ADP-ribosylation in the presence of pertussis toxin 0 allowed the detection of a great amount of ai subunit in the 0 E protein eluate of the affinity column, whereas no a3 protein * :3 was detectable in the purified receptor fraction (data not cd .; shown). b4 E 50 _ 0 DISCUSSION We describe in this report the purification of a pig brain galanin receptor essentially in a single step of ligand affinity chromatography. Thus, the unique strategy of direct synthe- - sis of the ligand on the hydrophiic polyacrylamide resin o 0 2 4 Bound recently designed to purify the vasoactive intestinal peptide /I A receptor from liver (18), proved useful to purify a galanin 7/X- 9 8 7 6 receptor. Two other key factors contribute to our purifica- - log [peptide] tion: (i) solubilization of galanin receptor in a nonaggregated FIG. 3. Competition between 1251-labeled galanin and unlabeled state with no loss of binding activity and (ii) differential galanin or its fragments for binding to the purified receptor. 1251- elution of the receptor and contaminating proteins from the labeled galanin was incubated for 16 h at 4°C with a constant amount affinity column by using acid pH elution. of magnesium acetate eluate and increasing concentrations of gala- This simple and rapid procedure gives a protein fraction nin-(1-29) (.), galanin-(2-29) (*), galanin-(3-29) (o), or galanin-(1- that binds galanin specifically and in a saturable manner with 15) (o). Results are expressed as percentage of specific binding a Kd value of 10 nM. The specific activity of the purified measured in the presence of tracer alone. For the galanin fragments, protein (17 nmol/mg of protein) was close to the theoretical results represent the mean ± SEM of three experiments. For value (18.5 nmol/mg of protein) for a 54-kDa galanin-(1-29), results represent the mean ± SEM of eight experi- protein binding ments. (Inset) Scatchard analysis of galanin binding. B/F values are one galanin molecule, suggesting that the receptor prepara- expressed x 10-2; bound values are expressed as nM. tion is homogenous. Downloaded by guest on September 30, 2021 Neurobiology: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3849 Thus, affinity chromatography results in the purification to purification procedure for galanin receptor should allow apparent homogeneity of a major protein band of 54 kDa as further studies on the molecular biology of this neuropeptide visualized by SDS/PAGE and silver staining or autoradio- receptor. Indeed, it is likely that the present procedure will graphy. This protein was identified as a galanin receptor by be valuable for the large-scale purification of galanin recep- affinity labeling with 1251-labeled galanin in the presence of tors required for microsequencing and cloning. DST. A molecular mass of -54 kDa has been also determined for the pig membrane and soluble galanin receptor under A.F. is a scientist of the Fonds de la Recherche en Sant6 du denaturing conditions by cross-linking of 1251-labeled gala- Qudbec. This work was supported by the Fondation pour la Recher- nin-receptor complexes and SDS/PAGE analysis (see Fig. che M6dicale (postdoctoral fellowship to Y.C.), the Association pour 1). In addition, most of the characteristics of galanin binding la Recherche sur le , and the Medical Research Council of to the membrane (29, 30) and CHAPS-solubilized (17) galanin Canada. receptor have been recovered with the purified galanin 1. Tatemoto, K., Rokaeus, A., Jomvall, H., McDonald, T. J. & Mutt, V. receptor, such as, the high peptide specificity of galanin (1983) FEBS Lett. 164, 124-128. binding and the structural requirement toward galanin and its 2. Ch'ng, J. L. C., Christofides, N. D., Anand, P., Gilson, S. J., Allen, fragments with the same rank order ofaffinities [i.e., galanin- Y. S., Su, H. C., Tatemoto, K., Morrison, J. F. B., Polak, J. M. & (1-29) > galanin-(2-29) > galanin-(1-15)J, whereas galanin- Bloom, S. R. (1985) Neurosciences 16, 343-354. (3-29) was not recognized by the purified receptor. 3. Hokfelt, T., Bartfai, T., Jacobowitz, D. & Ottoson, D. (1991) in Galanin: A New MultifunctionalPeptide in the Neuro-Endocrine System, Wenner- However, it must be pointed out that the purified galanin Gren International Symposium Series (Macmillan, Cambridge, U.K.), receptor differs from the membrane and CHAPS-soluble Vol. 58, pp. 199-211. forms by two important aspects: (i) its affinity for galanin (10 4. Fisone, G., Wu, C. F., Consolo, S., Nordstrom, O., Brynne, N., Bartfai, nM vs. 2.8 nM) and (ii) its lack of sensitivity to guanine T., Melander, T. & Hdkfelt, T. (1987) Proc. Natl. Acad. Sci. USA 84, of the lower affinity of the 7339-7343. nucleotides. An interpretation 5. Dunning, B. E. & Taborsky, G. J. (1988) Diabetes 37, 1157-1162. purified galanin receptor is the modification of the environ- 6. Amiranoff, B., Servin, A. L., Rouyer-Fessard, C., Couvineau, A., ment of the galanin receptor due to its selective retention Tatemoto, K. & Laburthe, M. (1987) Endocrinology 121, 284-289. from the crude soluble material to the resin. Another expla- 7. Amiranoff, B., Lorinet, A. M., Lagny-Pourmir, I. & Laburthe, M. (1988) nation may be provided with the uncoupling of the G1 protein Eur. J. Biochem. 177, 147-152. 8. Amiranoff, B., Lorinet, A. M. & Laburthe, M. (1989)J. Biol. Chem. 264, from the galanin receptor during the purification process. 20714-20717. Indeed, as attested by the inhibitory effect of guanine nucle- 9. Lagny-Pourmir, I., Amiranoff, B., Lorinet, A. M., Tatemoto, K. & otides on the interaction of galanin with the CHAPS- Laburthe, M. (1989) Endocrinology 124, 2635-2641. solubilized galanin receptor, the pig brain galanin receptor in 10. Amiranoff, B., Lorinet, A. M. & Laburthe, M. (1991) Eur. J. Biochem. 195, 459-463. solution is associated with a G protein. The probable disso- 11. Servin, A. L., Amiranoff, B., Rouyer-Fessard, C., Tatemoto, K. & ciation of the Gi protein from the galanin receptor during the Laburthe, M. (1987) Biochem. Biophys. Res. Commun. 144, 298-306. purification step may also explain the lower affinity of the 12. Fisone, G., Langel, U., Carlquist, M., Bergman, T., Consolo, S., purified galanin receptor toward its ligand, a feature observed Hokfelt, T., Und6n, A., Andell, S. & Bartfai, T. (1989) Eur. J. Biochem. 181, 269-276. for receptors associated with G protein (for review, see ref. 13. Chen, Y. H., Laburthe, M. & Amiranoff, B. (1992) Peptides 13, 339-341. 31). 14. De Weille, J. H., Schmid-Antomarchi, H., Fosset, M. & Lazdunski, M. Numerous factors may lead to the dissociation of the G1 (1988) Proc. Natl. Acad. Sci. USA 85, 1312-1316. protein from the galanin receptor during the purification 15. Dunne, M. J., Bullet, M. J., Li, G. D., Wollheim, C. B. & Peterson, 0. H. (1989) EMBO J. 8, 413-420. process. Thus, we suggest that dilution of the CHAPS- 16. Homaidan, F. R., Sharp, G. W. G. & Nowak, L. M. (1991) Proc. Nati. solubilized galanin receptor and of the detergent and/or the Acad. Sci. USA 88, 8744-8748. altered environment of the receptor during retention on the 17. Chen, Y. H., Couvineau, A., Laburthe, M. & Amiranoff, B. (1992) polyacrylamide resin and its elution by acid buffer may Biochemistry 31, 2415-2422. 18. Couvineau, A., Voisin, T., Gujarro, L. & Laburthe, M. (1990) J. Biol. contribute to the release of the galanin receptor from the G1 Chem. 265, 13386-13390. protein-galanin receptor complex. Although it is not a gen- 19. Forest, M., Martel, J.-C., St. Pierre, S., Quirion, R. & Fournier, A. (1990) eral feature, the physical uncoupling ofthe G protein from the J. Med. Chem. 33,1615-1619. peptide receptor during its purification was also reported to 20. Mitchell, A. R., Kent, S. B. H., Engelhard, M. & Merrifield, R. B. (1978) J. Org. Chem. 43, 2845-2852. occur in the course ofpurification ofthe gastric 21. Le-Nguyen, D., Heitz, A. & Castro, B. (1987) J. Chem. Soc. Perkins receptors (32) and the kidney receptors (33). Trans. 1, 1915-1919. In other studies, the coupling of the G protein with the 22. Van Wandelen, C., Zeikus, R. & Tsou, D. (1989) Chemistry Update peptide receptor may survive the purification procedure of (MilliGen/Biosearch, Novato, CA). 23. Wessel, D. & Fltlgge, U. I. (1984) Anal. Biochem. 138, 141-143. the receptor as described for the liver receptor 24. Laemmli, U. K. (1970) Nature (London) 227, 680-685. (34) and the lizard brain melatonin receptor (35). Occasion- 25. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310. ally, the purified receptor and the G protein, although dis- 26. Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194, 495- sociated during the purification process, may be copurified 496. 27. Bradford, M. (1976) Anal. Biochem. 72, 248-254. during their elution from the ligand affinity column as seen 28. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 197, 220-239. with the bovine brain adenosine receptor (36). In many 29. Lagny-Pourmir, I., Lorinet, A. M., Yanaihara, N. & Laburthe, M. (1989) instances, as for the pig liver vasoactive intestinal peptide Peptides 10, 757-761. receptor (18) or the releasing peptide receptor from 30. Amiranoff, B., Lorinet, A. M., Yanaihara, N. & Laburthe, M. (1989) Eur. J. Pharmacol. 163, 757-761. 3T3 cells (37), dissociation of the peptide receptor from G 31. Birnbaumer, L., Abramowitz, J. & Brown, A. M. (1990) Biochim. protein has even occurred earlier during the step ofdetergent Biophys. Acta 1031, 163-224. solubilization of the receptor. Whether these discrepancies 32. Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F. & Lewin, are related to the structure of receptors or to the various M. J. M. (1989) J. Biol. Chem. 264, 18789-18795. 33. Sheikh, S. P., Hansen, A. P. & Williams, J. A. (1991)J. Biol. Chem. 266, conditions used for their solubilization and purification re- 23959-23966. mains to be established. 34. Fishman, J. B., Dickey, B. F. & Fine, R. E. (1987) J. Biol. Chem. 262, In conclusion, the purification of a brain galanin receptor 14049-14055. as described in this is a 35. Rivkees, S. A., Conron, R. W. & Reppert, S. M. (1990) Endocrinology in an active form, paper, significant 127, 1206-1214. step toward the resolution of components involved in the 36. Munshi, R. & Linden, J. (1989) J. Biol. Chem. 264, 14853-14859. signal transduction pathway of this receptor, at least in the 37. Feldman, R. I., Wu, J. M., Jenson, J. C. & Mann, E. (1990) J. Biol. inhibition of adenylate cyclase (13). The availability of a Chem. 265, 17364-17372. Downloaded by guest on September 30, 2021