Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3845-3849, May 1993 Neurobiology Purification of a galanin 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 galanin receptor 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, glucagon, vasoactive showed a single polypeptide of 54 kDa by silver staining and intestinal peptide, synthetic neurotensin, substance P, baci- after radioiodination. Cross-linking of a purified fraction af- tracin, leupeptin, 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 insulin 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 neuropeptide 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 amino acid 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 central nervous system (3, 4) and a methyl (0.3 mmol/g), purchased sympathetic neuromodulator in the endocrine pancreas (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.