Proc. Natl. Acad. Sci. USAVol. 90, pp. 3845-3849, May 1993Neurobiology

Purification of a galanin receptor from pig brainYAOHUI 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 HenriHuchard-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 galanin receptor protein was solubilizedwith 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) from pig brain membranes and then pu-rified by single-step affinity chromatography. The productexhibits saturable and specific binding for galanin with abinding activity of 17 nmol/mg of protein and a dissociationconstant (Kd) of 10 nM. This represents a 300,000-fold puri-fication over the detergent-solubilized fraction with a finalrecovery of 31% of the initial membrane galanin bindingactivity. Gel electrophoresis of the affinity-purified materialshowed a single polypeptide of 54 kDa by silver staining andafter radioiodination. Cross-linking of a purified fraction af-rmity-labeled with 125I-labeled galanin revealed a single bandfor the galanin-receptor complex at 57 kDa. The generalbinding characteristics of the purified preparation appeared tobe identical to those of the crude soluble material as far asspecificity toward galanin and the structural requirement forgalanin are concerned. In contrast, unlike the CHAPS-solublegalanin receptor, binding of 125I-labeled galanin to the purifiedgalanin receptor was not sensitive to guanine nucleotides,suggesting that dissociation ofthe inhibitory guanine nucleotidebinding protein from the galanin receptor occurred duringpurification. The purification to homogeneity of a galaninreceptor paves the way toward its sequencing and cloning.

Galanin is an ubiquitous neuropeptide identified in porcineintestine on the basis of its amidated C terminus (1). Inagreement with its widespread localization in the central andperipheral nervous system (2), galanin elicits a wide range ofbiological responses (3). Since the discovery of galanin as aneuromodulator in the central nervous system (3, 4) and asympathetic neuromodulator in the endocrine pancreas (5),its mechanism of action in these organs has been the focus ofintensive study. Thus, radiolabeled galanin has been used toidentify specific galanin binding sites mostly in the endocrinepancreas (6-10) and brain (11, 12). In all these studies, thegalanin receptor was shown to be coupled to a guaninenucleotide binding regulatory (G) protein to initiate thecascade of cellular responses through inhibition of adenylatecyclase (7, 13), activation of ATP-sensitive K+ channels (14,15), or inhibition of calcium current (16).The molecular characterization of galanin receptors from

brain and pancreas by covalent labeling with 125I-labeledgalanin demonstrated that the galanin receptor is a mono-meric glycoprotein of 53-54 kDa (6, 8, 10, 11). Meanwhile,the functional association of the galanin receptor with aninhibitory G (Gi) protein was demonstrated (7, 9, 10, 12, 15).More recently, the solubilization of a rat brain galaninreceptor in an active form, a preliminary step toward itspurification, confirmed the physical association ofthe galaninreceptor in solution with the a subunit of a Gi protein (17).

In the present study, with the use of a one-step affinitychromatography (ref. 18 and A.F., A.C., and M.L., unpub-

lished data), we report the purification of a galanin receptorfrom pig brain, a rich source of receptors that is available inlarge amounts. This represents a basic step toward knowl-edge of the pharmacology and biochemistry of galanin re-ceptors and should lead to a better understanding of theirexpression in the organism.

METHODSMaterials. Synthetic porcine galanin, glucagon, vasoactive

intestinal peptide, synthetic neurotensin, substance P, baci-tracin, leupeptin, pepstatin A, GTP, GDP, guanosine 5'-[13,v-imido]triphosphate, cholesteryl hemisuccinate, 3-[(3-cholami-dopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),and phenylmethylsulfonyl fluoride were obtained from Sigma;porcine insulin was from Novo Research Institute (Copenha-gen); SDS/PAGE chemicals and the gel silver-staining kitwere from Bio-Rad; marker proteins were from BRL; disuc-cinimidyl tartarate (DST) was from Pierce; tert-butoxycar-bonyl(Boc)amino acids and benzotriazolyl-N-oxy-tris(dimeth-ylamino)phosphonium hexafluorophosphate (BOP) were fromRichelieu Biotechnologies (St-Hyacinthe, Quebec, Canada);fluoren-9-ylmethoxycarbonyl (Fmoc)-protected amino acidswere from Advanced ChemTech, and diisopropylethylamine(DIEA) was from Pfaltz & Bauer and was distilled fromninhydrin before use. Reagents used as scavengers duringdeprotection of the amino acid side chains (thioanisole,ethanedithiol, or anisole) were obtained from Aldrich. PepSynGel, a polyamide-based support functionalized with sarcosinemethyl ester residues (0.3 mmol/g), was purchased fromMilliGen (Mississauga, Ontario, Canada). Synthetic porcinegalanin was radioiodinated with 125I (Amersham) by the chlo-ramine-T method (26), at a specific activity of 700 Ci/mmol (1Ci = 37 GBq). Under those conditions, the tracer was prob-ably iodinated at the two tyrosine residues present in thepeptide. The porcine galanin fragments galanin-(2-29), gala-nin-(3-29), and galanin-(1-15) were kindly provided by N.Yanaihara (Shizuoka, Japan).

Solubilization of Galanin Receptors. Membranes from onepig brain were prepared and solubilized essentially as de-scribed for rat brain (11, 17). Briefly, for solubilizing thegalanin receptors, 30 ml of pig brain membrane suspension(7.5 mg/ml) was incubated in 20 mM Hepes buffer (pH 7.5)containing 30 mM CHAPS, 1.8 mM cholesteryl hemisucci-nate, 0.1 mM phenylmethylsulfonyl fluoride, 0.01% sodiumazide, 30% (vol/vol) glycerol, and 25 mM KCl for 30 min at0°C. The suspension was then diluted in 2 vol of the samebuffer but without CHAPS and centrifuged for 60 min at

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate; DST, disuccinimidyl tartarate; G protein, gua-nine nucleotide binding regulatory protein; Gi protein, inhibitory Gprotein; BOP, benzotriazolyl-N-oxy-tris(dimethylamino)phospho-nium hexafluorophosphate; Boc, tert-butoxycarbonyl; DIEA, diiso-propylethylamine; DMF, dimethyl formamide; Fmoc, fluoren-9-ylmethoxycarbonyl.TTo whom reprint requests should be addressed.

3845

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 29

, 201

9

Proc. Natl. Acad. Sci. USA 90 (1993)

100,000 x g (4°C). The supernatant was used as startingmaterial for further purification of galanin receptors.

Synthesis of the Galanin Matrix. The galanin matrix wasobtained by synthesizing the peptide on functionalized poly-acrylamide resin. A similar procedure was used to synthesizea hydrophobic affinity matrix for the purification of thevasoactive intestinal peptide receptor (A.F. et al., unpub-lished data).Resin preparation. The PepSyn Gel resin, formed by

copolymerization of dimethylacrylamide with a cross-linkingmonomer, was functionalized with sarcosine methyl ester(0.3 mmol/g). Prior to peptide synthesis, the support (2.4mmol, 8 g) was treated overnight with excess ethylenedi-amine (250-300 ml), which provided primary amine sites asattachment points. After the incorporation of ethylenedi-amine into the resin, Boc-E-aminocaproic acid (Boc-Aca), a6-carbon spacer arm, was coupled to the support by usingdimethyl formamide (DMF) as solvent. A 3-fold excess ofBoc-Aca (7.2 mmol, 1.67 g) was used for the coupling andactivation of the carboxylic function was achieved with BOPreagent (7.2 mmol, 3.18 g) in presence of DIEA (5-foldexcess, 12 mmol, 1.55 g, 2.1 ml) (19).Loading of the first amino acid. After equilibration of the

gel (0.6 mmol, 2 g) in methylene chloride, the Boc protectiongroup on aminocaproic acid was removed by 40% (vol/vol)trifluoroacetic acid in CH2Cl2 (one 5-min treatment and one20-min treatment), followed by successive washings withCH2Cl2 (two washes), isopropanol (two washes), and DMF(two washes). The C-terminal residue alanine was thenintroduced into the modified polyacrylamide resin as a Boc-alanyl-4-(oxymethyl)phenylacetic acid (OMPA) derivative.This compound was synthesized by the procedure of Mitchellet al. (20). Boc-Ala-OMPA (1.8 mmol, 0.61 g) was coupled tothe polymer by using the concomitant neutralization-coupling step strategy (19, 21) with BOP reagent (1.8 mmol,0.79 g) and DIEA (2.7 mmol, 0.35 g, 0.47 ml).Assembly of the peptide chain. Before being treated with

40% trifluoroacetic acid in CH2Cl2, the Boc-Ala-resin waswashed successively with DMF (two washes), isopropanol(two washes), and CH2Cl2 (two washes). Then, Boc-Leu28and Boc-Gly27 were incorporated in the peptide chain byfollowing the concomitant neutralization-coupling step pro-tocol (19, 21). After the deprotection of Gly27, the resin waswashed with CH2Cl2 (three washes) and incubated for 2 minwith 5% (vol/vol) DIEA in CH2Cl2. The resin was rinsed withCH2Cl2 (two rinses) and DMF (two rinses), and Fmoc-Tyr(butyl)26-COOH was coupled using four equivalents ofeach coupling reactants (Fmoc-amino acid, BOP, and DIEA).All the subsequent amino acid residues were introduced inthe peptide chain as Fmoc derivatives by using the neutral-ization-coupling step strategy with BOP reagent. Side-chainprotection of a-Fmoc-amino acids was as follows: Arg[4-methoxy-2,3,6-trimethylbenzene sulfonyl (Mtr)], Asn[triphenylmethyl (Trt)], Asp (O-butyl), His (Trt), Lys (Boc),Ser (butyl), Thr (butyl), and Tyr (butyl).Removal of the side-chain protecting groups. After com-

pletion of the synthesis, the peptide support was washed withisopropanol and methanol and dried overnight in vacuo. Apart of the resin (2.2 g) was transferred into a round-bottomflask. Then, 75 ml of reagent R (22) [90% (vol/vol) trifluoro-acetic acid/5% (wt/vol) thioanisole/3% (wt/vol) 1,2-ethanedithiol/2% (vol/vol) anisole] was added and the flaskwas purged with nitrogen. The flask was shaken for 7 h byusing a wrist-action shaker. The content of the flask waspoured in a fritted-glass filter and washed successively with50-100 ml of trifluoroacetic acid, isopropanol, and water.After a final washing step with isopropanol, the polymericsupport was dried 24 h in vacuo.

Affinity Chromatography of Solubilized Galanin Receptor.The galanin-(1-29)-polyacrylamide resin (100 mg, which con-

tained 49 mg of galanin) was pretreated with 40 ml of distilledwater for 48 h, packed into a plastic column (1 x 10 cm), andequilibrated with 20 ml of 20 mM Hepes buffer (pH 7.5)containing 10% (vol/vol) glycerol, 1 mM CHAPS, 0.12 mMcholesteryl hemisuccinate, 1 mM EDTA, 25 mM KCI, 20mMMgCl2, 0.01% sodium azide, and a protease inhibitor mixture[1 mM phenylmethylsulfonyl fluoride/leupeptin (10 mg/liter)/pepstatin A (10 mg/liter)] (buffer A). Soluble material(30 ml) containing active galanin receptor (-3.8 mg ofproteinper ml) was loaded on the affinity column at a flow rate of 12ml/h at 4°C and recycled overnight through the column. Atthe end of the recycling phase, the pass-through fraction,which contained no binding activity, was collected and storedat -80°C. The resin was then washed with 100 ml of bufferA until the eluent was protein-free. The galanin receptorsbound to the affinity gel were eluted with 36 ml of 10 mMmagnesium acetate, pH 4.0/10% glycerol/0.01% sodiumazide and the protease inhibitor mixture at a flow rate of 30ml/h. The fractions containing galanin receptors were im-mediately neutralized with 2 M Hepes and used for ligandbinding or stored at -80°C. After each purification proce-dure, the affinity column was washed successively with 5 volof 1 M NaCl, 5 vol of 0.2 M acetic acid, 5 vol of 20% ethanol,and 10 vol of 0.04% sodium azide in H20. The affinity gel wasused immediately for another purification cycle or stored at4°C. It could be used for at least five successive purificationprocedures without any detectable modification.

Binding and Cross-Linking of 12II-Labeled Galanin to Mem-branes and Soluble and Purified Receptors. Assays of bindingto membrane and solubilized galanin receptor were carriedout as described (11, 17). For the purified receptor, 150 ,lI offractions eluted from the affinity column and containing thebinding activity was incubated with 50 ,ul of the pass-throughfraction, which signiflcantly increased galanin binding activ-ity (see above), and 0.5 nM 125I-labeled galanin for 16 h at 4°Cin a final volume of 250 ,ul of 20 mM Hepes buffer (pH 7.5)containing 2% (wt/vol) bovine serum albumin, bacitracin (1mg/ml), and the protease inhibitor mixture. The reaction wasterminated by fitration through Whatman GF/C glass fiberfilters pretreated with 0.5% polyethylenimine in water.

Affinity cross-linking of 125I-labeled galanin bound to mem-brane or soluble receptors was performed as described (11,17). Cross-linking of 125I-labeled galanin to the purified re-ceptors (0.3 ,ug of protein per ml) was performed by incu-bating the purified galanin receptor with the tracer in 20 mMHepes buffer (pH 7.5) for 16 h at 40C in the presence orabsence of 1 AM unlabeled galanin. The 125I-labeled galanin-receptor complexes were then cross-linked by addition ofDST at a final concentration of 1 mM for 20 min at 4°C andthe reaction was stopped by adding 20 ,ul of ice-cold 1 MTris HCl (pH 6.8). The proteins were then precipitated witha mixture of a methanol/chloroform/water, 4:1:3 (vol/vol),as described by Wessel and Flugge (23). Precipitates wereredissolved in SDS sample buffer (pH 6.8) containing 10%glycerol, 3% (wt/vol) SDS, and 0.001% bromophenol blue,boiled for 3 min at 100°C, and analyzed by SDS/PAGE.SDS/PAGE. Gel electrophoresis was performed, as de-

scribed by Laemmli (24), using a 5% polyacrylamide stackinggel and a 12% polyacrylamide slab gel as described (8). Thegels were stained, dried, and exposed to a Trimax type XMfilm (3M) with a 3M Trimax intensifying screen for 1-7 daysat -80°C. The following proteins of known molecular masswere used to calibrate the gels: myosin (200 kDa), phosphor-ylase b (92 kDa), bovine serum albumin (68 kDa), ovalbumin(43 kDa), and carbonic anhydrase (29 kDa). Silver staining ofgels was performed as described by Morrissey (25).

lodination of the Purified Receptor. The purified receptoreluted from the affinity column was iodinated by the chlora-mine-T method (26). Briefly, 3 ml of the fractions containingthe galanin binding activity (300 ng) was precipitated with 24

3846 Neurobiology: Chen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 29

, 201

9

Proc. Natl. Acad. Sci. USA 90 (1993) 3847

ml of methanol/chloroform/water, 4:1:3 (vol/vol). The pre-

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 presenceof 100 ,ug of chloramine T for 1 min at room temperature (1Ci = 37 GBq). The reaction was stopped by adding 200 ,g ofsodium metabisulfite. The iodinated receptor was separatedfrom free 125I on a Sephadex G-50 column, using 0.2 M aceticacid containing 0.5% bovine serum albumin and 0.03% bac-itracin. The radioiodinated receptor was precipitated andsubmitted to SDS/PAGE.

ADP-Ribosylation of Fractions Eluted from the GalaninAffinity Chromatography Column. ADP-ribosylation withpertussis toxin was carried out essentially as described (10).Briefly, 1 ml of material was incubated with preactivatedpertussis toxin (100 ng/ml) in 5 mM Tris-HCl (pH 7.5)containing 1 mM ATP, 100 AM GTP, 2.5 mM MgCl2, 1 mMdithiothreitol, and 10 ,uM [32P]NAD. After incubation for 15min at 30°C, the proteins were precipitated with methanol/chloroform/water, 4:1:3 (vol/vol), and redissolved in SDSsample buffer (pH 6.8) before analysis by SDS/PAGE.

Protein Determination. Protein concentration ofmembraneand soluble material was measured using the procedure ofBradford (27) with bovine serum albumin as standard. Pro-teins in the eluted fractions were determined after SDS/PAGE by densitometric scanning of silver-stained gels usingthe standard molecular weight markers (see above).Data Analysis. Analysis of saturation and competition of

galanin binding experiments was performed by the LIGANDprogram (28).

RESULTS

Membrane and Soluble Galanin Receptors. The character-istics of galanin receptors from membranes of pig braincompared well with those from rat brain (11, 12, 29). (i)Scatchard analysis of binding data indicated the existence ofone type of high-affinity site (Kd = 0.76 nM) with a Bm., of55 fmol/mg of protein (Table 1). (ii) The galanin fragmentsgalanin-(2-29) and galanin-(1-15) competitively inhibitedbinding of 125I-labeled galanin to membranes whereas gala-nin-(3-29) was inactive (data not shown). (iii) The binding ofgalanin was inhibited by guanine nucleotides in a dose-dependent manner and with the following rank order ofpotency: guanine 5'-[3,y,-imido]triphosphate > GTP > GDP.The nucleotides caused an 80% reduction in the binding of125I-labeled galanin and the IC50 values were 20 ,uM, 0.1 mMand 0.2 mM, respectively (data not shown). (iv) Cross-linkingexperiments showed that the galanin-receptor complex ofpigbrain membranes behaved as a protein of 57 kDa, the labelingof which was abolished by an excess of native galanin (Fig.1, lane A).

Solubilization of active galanin receptors from pig brainwas achieved using the following optimal conditions: 30 mMCHAPS and 7.5 mg of protein per ml. The solubilized galaninreceptors bound galanin with a high affinity (Kd = 2.78 nM)and a Bm,, of 56 fmol/mg of protein (Table 1). The solublegalanin receptor retained its sensitivity to the inhibitoryeffects of guanine nucleotides (data not shown). Cross-linking of 125I-labeled galanin to the CHAPS-solubilized pigbrain galanin receptor showed that the soluble galanin-

A B C

kDa

200 -

92.5 -

68 -

57 -

43 -

29 -

Gal - + +I +

FIG. 1. Autoradiography of cross-linked 125I-labeled galanin tomembrane, CHAPS-solubilized, and purified pig brain galanin re-ceptors. Membranes (lanes A), CHAPS-solubilized extracts (lanesB), and purified galanin receptors (lanes C) were incubated with1251-labeled galanin (0.5 nM) in the absence (-) or presence (+) of 1,uM unlabeled galanin (Gal). DST (1 mM) was added to the samplesthat were further analyzed by SDS/PAGE. The molecular mass ofthe galanin-receptor protein complex was estimated at 57 kDa.

receptor complex behaved as a protein of57 kDa, the labelingof which was specific for galanin (Fig. 1, lane B).

Receptor Purification. The data obtained from five receptorpurification experiments are summarized in Table 1. Thesolubilized material (30 ml of CHAPS-solubilized extract)was allowed to bind overnight to the galanin-polyacrylamideresin. This overnight recycling completely removed the 1251_labeled galanin binding activity in the extract. In contrast,only 3% of the protein was adsorbed on the resin. Most ofthese proteins were eluted from the column after extensivewashing with buffer A. No galanin binding activity was foundin this eluted fraction (Fig. 2). Magnesium acetate (pH 4) wasused to eluate the galanin receptor from the column in viewof the previous observation that the galanin receptor issensitive to pH changes and that a slightly acid buffer greatlyreduces the affinity of the receptor for agonists (9). The totalamount of protein eluted from the column in fractions con-taining 125I-labeled galanin binding activity was estimatedfrom the trace of the A280 of the sample. After neutralization,the binding of 125I-labeled galanin to the purified materialeluted from the affinity column was found to be specific andsaturable (Fig. 3). Parameters obtained from the correspond-ing Scatchard plot (Fig. 3 Inset) were Kd = 10 nM and Bmax= 3 nM. From the amount of proteins estimated by silverstaining, the binding capacity ofthe putative galanin receptorwas calculated to be 17 nmol/mg of protein, which corre-sponds to an enrichment of 300,000 over the crude solublereceptor. This value is close to the theoretical value of 18.5nmol/mg calculated on the basis of one galanin binding siteper 54-kDa protein (Fig. 3 and Table 1). The yield of 125I1labeled galanin binding activity recovered from the columnwas =60% of the total activity loaded (Table 1).The ligand specificity of the purified galanin receptor was

investigated by analyzing the ability of various peptides tocompete with 1251-labeled galanin binding. Binding to themagnesium acetate eluate was specific to galanin, since it wasnot displaced by structurally unrelated peptides such as the

Table 1. Binding parameters of the pig brain galanin receptorTotal protein, Kd, Specific activity, Fold Recovery,

Preparation mg nM fmol/mg of protein purification tMembrane 225 0.76 55 1 100Crude soluble 114 2.78 56 1 52Purified 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.

Neurobiology: Chen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 29

, 201

9

Proc. Natl. Acad. Sci. USA 90 (1993)

01 0

.

iOE"0 X,b

la4-

Fraction number

FIG. 2. Affinity chromatography of CHAPS-solubilized galaninreceptor. Soluble proteins (30 ml) were loaded at 4°C on the galanin-polyacrylamide resin column (1 x 10 cm) equilibrated with buffer A.The column was washed with buffer A (fractions 1-50) and subse-quently with 36 ml of elution buffer containing 10 mM magnesiumacetate (pH 4), 10%o glycerol, 0.01% sodium azide, and the proteaseinhibitor mixture (fractions 51-65). Chromatography was followedby automatic recording of A28o (A) and by measuring the specificbinding of 1251-labeled galanin (0.1 nM) to aliquots of each fraction(o). Fraction volume, 2 ml; flow rate, 30 ml/h.

vasoactive intestinal peptide, glucagon, insulin, substance P,or neurotensin (data not shown).The fractions containing 1251-labeled galanin binding activ-

ity were pooled, concentrated, and analyzed by SDS/PAGE(Fig. 4, lane B). The gel showed presence of a single band ofsilver-stained material with a molecular mass of 54 kDa,indicating that the protein is free of significant contaminants.The 54-kDa protein was purified with the galanin affinitycolumn, as shown by the profile of proteins present in thesolubilized brain tissue before affinity chromatography. In-deed, Fig. 4, lane A, shows the electrophoretic patternobtained with the crude CHAPS extract including a largenumber of protein bands with molecular masses from >100 to<10 kDa. The direct radioiodination of the purified receptor

100

'000 E* :3

cd .;b4 E

0

50 _

o

7/X- 9 8 7- log [peptide]

6

FIG. 3. Competition between 1251-labeled galanin and unlabeledgalanin or its fragments for binding to the purified receptor. 1251-labeled galanin was incubated for 16 h at 4°C with a constant amountof magnesium acetate eluate and increasing concentrations of gala-nin-(1-29) (.), galanin-(2-29) (*), galanin-(3-29) (o), or galanin-(1-15) (o). Results are expressed as percentage of specific bindingmeasured in the presence of tracer alone. For the galanin fragments,results represent the mean ± SEM of three experiments. Forgalanin-(1-29), results represent the mean ± SEM of eight experi-ments. (Inset) Scatchard analysis of galanin binding. B/F values areexpressed x 10-2; bound values are expressed as nM.

kDa

29 v2..

A

FIG. 4. Electrophoretic analysisof soluble and purified receptor pro-tein. Lanes: A, crude CHAPS extract(10 j.g of protein) was submitted di-rectly to SDS/PAGE and the gel was

MM _w silver-stained; B, 300 ng of the recep-tor preparation was electrophoreti-cally resolved and visualized by silverstaining; C, autoradiography of theiodinated galanin receptor after expo-sure of gel to a 3M Trimax intensify-

B C ing screen for 12 h at -80°C.

revealed a single radioactive band at 54 kDa (Fig. 4, lane C),confirming the homogeneity of the purified preparation.

Additional experiments were conducted, supporting theconclusion that the 54-kDa protein is a galanin receptor. Theneutralized magnesium acetate-eluted fractions were incu-bated with 1251-labeled galanin in the absence and in thepresence ofan excess ofnative galanin. The cross-linker DST(1 mM) was added to the medium, which was analyzed bySDS/PAGE. Fig. 1, lane C, shows a single labeled band at 57kDa. This band was not observed when incubation wasperformed in the presence of 1 ,uM native galanin (Fig. 1, laneC). If one molecule of 125I-labeled galanin (3 kDa) is boundper molecule of receptor, the intrinsic molecular mass of thepurified galanin receptor is estimated at 54 kDa by affinitylabeling experiments.The relative affinities of the fragments galanin-(2-29),

galanin-(3-29), and galanin-(1-15) for the purified galaninreceptor (Fig. 3), the membrane receptor (28), and theCHAPS-solubilized galanin receptor in crude extracts (17)were similar, indicating that the structural requirement of thereceptor for binding its ligand was not altered by purification.However, the sensitivity to guanine nucleotides of the

purified galanin receptor was altered. Indeed, the binding of125I-labeled galanin to the purified extract was unaffected bythese compounds, indicating that the purified receptor doesnot maintain its connection to its G protein. Attempts toidentify the inhibitory a; subunit of the inhibitory G, proteinin the different fractions eluted from the affinity column by[32P]ADP-ribosylation in the presence of pertussis toxinallowed the detection of a great amount of ai subunit in theprotein eluate of the affinity column, whereas no a3 proteinwas detectable in the purified receptor fraction (data notshown).

DISCUSSIONWe describe in this report the purification of a pig braingalanin receptor essentially in a single step of ligand affinitychromatography. Thus, the unique strategy of direct synthe-sis of the ligand on the hydrophiic polyacrylamide resinrecently designed to purify the vasoactive intestinal peptidereceptor from liver (18), proved useful to purify a galaninreceptor. Two other key factors contribute to our purifica-tion: (i) solubilization of galanin receptor in a nonaggregatedstate with no loss of binding activity and (ii) differentialelution of the receptor and contaminating proteins from theaffinity column by using acid pH elution.

This simple and rapid procedure gives a protein fractionthat binds galanin specifically and in a saturable manner witha Kd value of 10 nM. The specific activity of the purifiedprotein (17 nmol/mg of protein) was close to the theoreticalvalue (18.5 nmol/mg of protein) for a 54-kDa protein bindingone galanin molecule, suggesting that the receptor prepara-tion is homogenous.

- 0 2 4Bound

/I A

3848 Neurobiology: Chen et al.

s

Dow

nloa

ded

by g

uest

on

Dec

embe

r 29

, 201

9

Proc. Natl. Acad. Sci. USA 90 (1993) 3849

Thus, affinity chromatography results in the purification toapparent homogeneity of a major protein band of 54 kDa asvisualized by SDS/PAGE and silver staining or autoradio-graphy. This protein was identified as a galanin receptor byaffinity labeling with 1251-labeled galanin in the presence ofDST. A molecular mass of -54 kDa has been also determinedfor the pig membrane and soluble galanin receptor underdenaturing conditions by cross-linking of 1251-labeled gala-nin-receptor complexes and SDS/PAGE analysis (see Fig.1). In addition, most of the characteristics of galanin bindingto the membrane (29, 30) and CHAPS-solubilized (17) galaninreceptor have been recovered with the purified galaninreceptor, such as, the high peptide specificity of galaninbinding and the structural requirement toward galanin and itsfragments with the same rank order of affinities [i.e., galanin-(1-29) > galanin-(2-29) > galanin-(1-15)J, whereas galanin-(3-29) was not recognized by the purified receptor.However, it must be pointed out that the purified galanin

receptor differs from the membrane and CHAPS-solubleforms by two important aspects: (i) its affinity for galanin (10nM vs. 2.8 nM) and (ii) its lack of sensitivity to guaninenucleotides. An interpretation of the lower affinity of thepurified galanin receptor is the modification of the environ-ment of the galanin receptor due to its selective retentionfrom the crude soluble material to the resin. Another expla-nation may be provided with the uncoupling of the G1 proteinfrom the galanin receptor during the purification process.Indeed, as attested by the inhibitory effect of guanine nucle-otides on the interaction of galanin with the CHAPS-solubilized galanin receptor, the pig brain galanin receptor insolution is associated with a G protein. The probable disso-ciation of the Gi protein from the galanin receptor during thepurification step may also explain the lower affinity of thepurified galanin receptor toward its ligand, a feature observedfor receptors associated with G protein (for review, see ref.31).Numerous factors may lead to the dissociation of the G1

protein from the galanin receptor during the purificationprocess. Thus, we suggest that dilution of the CHAPS-solubilized galanin receptor and of the detergent and/or thealtered environment of the receptor during retention on thepolyacrylamide resin and its elution by acid buffer maycontribute to the release of the galanin receptor from the G1protein-galanin receptor complex. Although it is not a gen-eral feature, the physical uncoupling of the G protein from thepeptide receptor during its purification was also reported tooccur in the course of purification of the gastric somatostatinreceptors (32) and the kidney neuropeptide Y receptors (33).In other studies, the coupling of the G protein with thepeptide receptor may survive the purification procedure ofthe receptor as described for the liver vasopressin receptor(34) and the lizard brain melatonin receptor (35). Occasion-ally, the purified receptor and the G protein, although dis-sociated during the purification process, may be copurifiedduring their elution from the ligand affinity column as seenwith the bovine brain adenosine receptor (36). In manyinstances, as for the pig liver vasoactive intestinal peptidereceptor (18) or the gastrin releasing peptide receptor from3T3 cells (37), dissociation of the peptide receptor from Gprotein has even occurred earlier during the step of detergentsolubilization of the receptor. Whether these discrepanciesare related to the structure of receptors or to the variousconditions used for their solubilization and purification re-mains to be established.

In conclusion, the purification of a brain galanin receptorin an active form, as described in this paper, is a significantstep toward the resolution of components involved in thesignal transduction pathway of this receptor, at least in theinhibition of adenylate cyclase (13). The availability of a

purification procedure for galanin receptor should allowfurther studies on the molecular biology of this neuropeptidereceptor. Indeed, it is likely that the present procedure willbe valuable for the large-scale purification of galanin recep-tors required for microsequencing and cloning.

A.F. is a scientist of the Fonds de la Recherche en Sant6 duQudbec. This work was supported by the Fondation pour la Recher-che M6dicale (postdoctoral fellowship to Y.C.), the Association pourla Recherche sur le Cancer, and the Medical Research Council ofCanada.

1. Tatemoto, K., Rokaeus, A., Jomvall, H., McDonald, T. J. & Mutt, V.(1983) FEBS Lett. 164, 124-128.

2. Ch'ng, J. L. C., Christofides, N. D., Anand, P., Gilson, S. J., Allen,Y. S., Su, H. C., Tatemoto, K., Morrison, J. F. B., Polak, J. M. &Bloom, S. R. (1985) Neurosciences 16, 343-354.

3. Hokfelt, T., Bartfai, T., Jacobowitz, D. & Ottoson, D. (1991) in Galanin:A New Multifunctional Peptide in the Neuro-Endocrine System, Wenner-Gren International Symposium Series (Macmillan, Cambridge, U.K.),Vol. 58, pp. 199-211.

4. Fisone, G., Wu, C. F., Consolo, S., Nordstrom, O., Brynne, N., Bartfai,T., Melander, T. & Hdkfelt, T. (1987) Proc. Natl. Acad. Sci. USA 84,7339-7343.

5. Dunning, B. E. & Taborsky, G. J. (1988) Diabetes 37, 1157-1162.6. Amiranoff, B., Servin, A. L., Rouyer-Fessard, C., Couvineau, A.,

Tatemoto, K. & Laburthe, M. (1987) Endocrinology 121, 284-289.7. Amiranoff, B., Lorinet, A. M., Lagny-Pourmir, I. & Laburthe, M. (1988)

Eur. J. Biochem. 177, 147-152.8. Amiranoff, B., Lorinet, A. M. & Laburthe, M. (1989)J. Biol. Chem. 264,

20714-20717.9. Lagny-Pourmir, I., Amiranoff, B., Lorinet, A. M., Tatemoto, K. &

Laburthe, M. (1989) Endocrinology 124, 2635-2641.10. Amiranoff, B., Lorinet, A. M. & Laburthe, M. (1991) Eur. J. Biochem.

195, 459-463.11. Servin, A. L., Amiranoff, B., Rouyer-Fessard, C., Tatemoto, K. &

Laburthe, M. (1987) Biochem. Biophys. Res. Commun. 144, 298-306.12. Fisone, G., Langel, U., Carlquist, M., Bergman, T., Consolo, S.,

Hokfelt, T., Und6n, A., Andell, S. & Bartfai, T. (1989) Eur. J. Biochem.181, 269-276.

13. Chen, Y. H., Laburthe, M. & Amiranoff, B. (1992) Peptides 13, 339-341.14. De Weille, J. H., Schmid-Antomarchi, H., Fosset, M. & Lazdunski, M.

(1988) Proc. Natl. Acad. Sci. USA 85, 1312-1316.15. Dunne, M. J., Bullet, M. J., Li, G. D., Wollheim, C. B. & Peterson,

0. H. (1989) EMBO J. 8, 413-420.16. Homaidan, F. R., Sharp, G. W. G. & Nowak, L. M. (1991) Proc. Nati.

Acad. Sci. USA 88, 8744-8748.17. Chen, Y. H., Couvineau, A., Laburthe, M. & Amiranoff, B. (1992)

Biochemistry 31, 2415-2422.18. Couvineau, A., Voisin, T., Gujarro, L. & Laburthe, M. (1990) J. Biol.

Chem. 265, 13386-13390.19. Forest, M., Martel, J.-C., St. Pierre, S., Quirion, R. & Fournier, A. (1990)

J. Med. Chem. 33,1615-1619.20. Mitchell, A. R., Kent, S. B. H., Engelhard, M. & Merrifield, R. B.

(1978) J. Org. Chem. 43, 2845-2852.21. Le-Nguyen, D., Heitz, A. & Castro, B. (1987) J. Chem. Soc. Perkins

Trans. 1, 1915-1919.22. Van Wandelen, C., Zeikus, R. & Tsou, D. (1989) Chemistry Update

(MilliGen/Biosearch, Novato, CA).23. Wessel, D. & Fltlgge, U. I. (1984) Anal. Biochem. 138, 141-143.24. Laemmli, U. K. (1970) Nature (London) 227, 680-685.25. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310.26. Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194, 495-

496.27. Bradford, M. (1976) Anal. Biochem. 72, 248-254.28. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 197, 220-239.29. Lagny-Pourmir, I., Lorinet, A. M., Yanaihara, N. & Laburthe, M. (1989)

Peptides 10, 757-761.30. Amiranoff, B., Lorinet, A. M., Yanaihara, N. & Laburthe, M. (1989)

Eur. J. Pharmacol. 163, 757-761.31. Birnbaumer, L., Abramowitz, J. & Brown, A. M. (1990) Biochim.

Biophys. Acta 1031, 163-224.32. Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F. & Lewin,

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,

23959-23966.34. Fishman, J. B., Dickey, B. F. & Fine, R. E. (1987) J. Biol. Chem. 262,

14049-14055.35. Rivkees, S. A., Conron, R. W. & Reppert, S. M. (1990) Endocrinology

127, 1206-1214.36. Munshi, R. & Linden, J. (1989) J. Biol. Chem. 264, 14853-14859.37. Feldman, R. I., Wu, J. M., Jenson, J. C. & Mann, E. (1990) J. Biol.

Chem. 265, 17364-17372.

Neurobiology: Chen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 29

, 201

9