Cloning and Expressional Characterization of a Novel Galanin Receptor

Galanin, a 29–30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors.125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (K d = 0.6 nm) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin.

Galanin is a 29 -30 amino acid neuropeptide with no significant homology to any known family of biologically active peptides (1). Galanin is widely distributed in the central and peripheral nervous systems and is highly expressed in various regions of the brain. Many physiological processes are modulated by galanin, including neurotransmitter and hormone release (2), spinal reflexes, nociception (3), firing of noradrenergic neurons, and contraction of gastrointestinal smooth muscle (4,5). Like neuropeptide Y, centrally administered galanin potently stimulates food intake in animals (6), suggesting a role for galanin in control of body weight.
A large body of evidence suggests that galanin mediates the various physiological functions through interaction with distinct receptor subtypes. Pharmacological studies with several peptidic agonists and antagonists of galanin receptor suggest the existence of multiple receptor subtypes (7)(8)(9). The first of these receptors (GalR1) has been cloned from several species (10 -13). More recently, a second galanin receptor subtype (GalR2) was cloned. GalR2 is markedly dissimilar to the GalR1 receptor, sharing only 40% sequence homology (14,15). Hydropathy analysis suggests that both GalR1 and GalR2 receptors have seven hydrophobic transmembrane domains, typical of members of the G protein-coupled receptor superfamily (16). The GalR2 receptor is distinguished pharmacologically from the GalR1 receptor by its high affinity for ligand galanin- . In addition to the pharmacological differences, the GalR2 transcript is widely distributed in both central and peripheral tissues (14,15), whereas the expression of GalR1 is more restricted to brain and spinal cord (12,13).
Given that a large number of physiological actions are modulated by galanin, it is unlikely that the two cloned galanin receptors mediate all the functions of galanin. A complete understanding of the roles of galanin requires identification and characterization of all the galanin receptor subtypes. In this report, we describe the cloning of a novel rat galanin receptor subtype by polymerase chain reaction (PCR), 1 sib selection and rapid amplification of cDNA ends (RACE). The receptor shares highest homology with GalR1 and GalR2 and is capable of binding galanin and galanin analogs. We thus designate this new receptor as the GalR3 galanin receptor.
PCR Sib Selection-PCR with galanin receptor-specific primers was used to screen pools of a rat hypothalamus cDNA library. Positive pools so identified were subdivided and screened further by the PCR screening until the final round of screening, at which time individual colonies were selected by analysis of plasmid mini-preps. The rat cDNA library was constructed as described previously (15).
PCR and RACE Amplification of cDNA Fragment-Unless otherwise specified, PCR was performed using KlenTaq polymerase, which possesses proof reading activity (CLONTECH) and a cycling profile of 94°C for 1 min, 65°C for 1 min, and 72°C for 2 min (40 cycles). Approximately 1 l of overnight Escherichia coli culture was used in the PCR for sib selection. For RACE, nested primers specific to rat GalR3 cDNA and nested adaptor primers were used in the primary and secondary PCRs. A thermal cycling profile of 94°C for 30 s, 65°C for 30 s, and 72°C for 90 s (25 cycles) and rat brain cDNA as a template were used in primary PCR. A cycling profile of 94°C for 1 min and 70°C for 4 min (30 cycles) and 5 l of the primary PCR product (diluted 1:50) as a template were used in the secondary PCR. The RACE product was cloned into * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
DNA Sequencing and Analysis-The DNA sequences of clones were determined on both strands using Applied Biosystems Prism dye termination DNA sequencing reagents and an Applied Biosystems 373 automated sequencing apparatus (Perkin-Elmer). DNA and protein sequence comparisons were performed with the DNA* software (DNAstar Inc., Madison, WI).
Transfection of COS-7 Cells-Rat GalR3 cDNA was introduced into COS-7 cells by electroporation as described previously (15) or by the use of LipofectAMINE method (Life Technologies, Inc.) according to the manufacturer's instructions. The two methods gave comparable levels of expression.
Receptor Membrane Preparation-Three days following the transfection of the COS-7 cells, receptor membrane was prepared as previously described (15). 125 I-Galanin Binding Assay-Binding of 125 I-porcine galanin to the membrane preparations was performed as previously described (15) except that 20 g of membrane protein was used in the saturation and competition binding assays. All data were analyzed by nonlinear regression (Prism, GraphPad, San Diego, CA) and the K i calculated according to the method of Cheng and Prusoff (17).
Northern Blot Analysis of Rat GalR3-A rat multitissue Northern blot (CLONTECH) was hybridized for 15 h at 55°C in an ExpressHyb solution (CLONTECH) using 32 P-labeled rat GalR3 cDNA as a probe (labeled with a random priming kit, Life Technologies, Inc., specific activity ϭ 3 ϫ 10 9 cpm/g). After hybridization, the blot was washed with wash solution I (2 ϫ SSC, 0.05% SDS) for 30 min at room temperature then with wash solution II (0.1 ϫ SSC, 0.1% SDS) for 30 min at room temperature, 1 h at 48°C, 1 h at 52°C, and 30 min at 54°C. The blot was then wrapped with Saran Wrap and exposed to Kodak BioMax films at Ϫ80°C for 1 week. The same blot was stripped and hybridized in a similar manner with a 32 P-labeled actin cDNA to ensure loading of poly(A) ϩ mRNA from the tissues onto the blot.

RESULTS AND DISCUSSION
BLAST search (18) of the GenBank™ data base with the human GalR1 receptor amino acid sequence (10) as query identified a portion of a human genomic clone (accession number Z82241) that possessed high homology with the amino acids 37-132 of human GalR1 (third reading frame of the positive strand, 55% identity). The homology was greater than that between rGalR1 and rGalR2 (40%), suggesting that this human clone may contain a portion of a new galanin receptor.
Several pairs of PCR primers were generated according to the human genomic sequence and used in PCR with cDNA reverse-translated from rat liver RNA as a template to obtain rat GalR3 cDNA (Fig. 1A). A PCR cycling paradigm employing low annealing temperature with two PCR primers, oligonucleotide 93C and oligonucleotide 120B, produced a PCR product. The approximately 700-base pair fragment was cloned into vector pcR3.1 and sequenced. Comparison of the DNA sequence with the nucleotide sequences in GenBank™ revealed 86, 65, and 63% identities with the human genomic clone (Z82241), rat GalR2, and human GalR1, respectively. Therefore, the rat clone appeared to be a portion of a novel rat galanin receptor (Fig. 1A).
RACE and PCR sib selection were performed to extend the cDNA sequence toward the 5Ј and 3Ј directions. In RACE, primers oligo172 and AP1 (outer adaptor primer) were used in the primary PCR with a rat brain cDNA library as a template and primers oligo177 and AP2 (inner adaptor primer) were used in the secondary PCR with product of the primary PCR as for 0.5 min, and 72°C for 1 min (40 cycles) was used to obtain the 0.7-kb PCR product and standard cycling was used in other PCRs (see "Materials and Methods"). B, nucleotide sequence and the deduced amino acid sequence of the rat GalR3 receptor (GenBank™ accession number AF031522). The seven putative TMs are underlined. C, alignment of the amino acid sequence of rat GalR3 receptor with rat GalR1 and GalR2 (11,15). *, potential N-linked glycosylation site; #, two Cys residues that may form a disulfide bond. Gaps (-) are introduced to optimize the alignment. Conserved amino acid residues are shaded. The seven TMs are underlined. a template. The final RACE product, ϳ1.8-kb, overlapped with the 5Ј portion of the rat 0.7-kb GalR3 cDNA and the upstream 5Ј-untranslated region (Fig. 1A). For PCR sib selection, two primers, oligo164 and oligo167, were used to screen a cDNA library constructed from rat hypothalamus (15). Pool A28 gave a positive band and was subdivided and screened until a single clone (clone A28-1, 1.3 kb) was obtained (Fig. 1A).
A full-length clone of GalR3 cDNA was obtained by performing further sib selection on the hypothalamus cDNA library using primer oligo185, designed based on the sequence of the 5Ј-RACE product, and primer oligo184, designed based on the sequence of clone A28-1. A single clone A5-3, selected from library pool A5, was obtained and sequenced. The clone was 2.2 kb long and contained all the sequence of clone A28-1 and a portion of the 5Ј-RACE product (Fig. 1A). A complete open reading frame was identified corresponding to a protein of 370 amino acids with a calculated molecular mass of 40.3 kDa (Fig.  1B). The clone was termed GalR3 receptor. Hydrophathy analysis revealed seven putative transmembrane-spanning domains (TMs) typical of G protein-coupled receptors. The GalR3 receptor also contains a single potential N-linked glycosylation site in the N-terminal region, two Cys residues in the first and second extracellular loops that form a putative disulfide bond in these receptors, and two Cys residues in the C-terminal region that may be involved in palmitoylation (Fig. 1C).
The amino acid sequence of the rat GalR3 receptor is significantly different from those of rat GalR1 and GalR2. The overall homology is 36% to rat GalR1 and 54% to rat GalR2 as analyzed by the Jutun Hein method (19) (Fig. 1C). A search of the SwissProt data bank revealed that rat GalR3 has high homology to the rat somatostatin type 4 receptor (31%) (20) and the rat -type opioid receptor (27%) (21). Sequence alignment of rGalR3 with rGalR1 shows the greatest similarity in TM2, TM7, and TM1, being 62, 46, and 46% identical, respectively. Alignment with rGalR2 displayed a generally higher homology in the TMs than rGalR1, with the highest in TM2, TM3, and TM4, being 92, 83, and 71% identical, respectively. The N terminus and the C terminus possess least homology with both rGalR1 and rGalR2 receptors (Fig. 1C).
Northern blot analysis was performed to examine the tissue distribution of GalR3 mRNA. A single band of ϳ3.5 and ϳ3 kb was detected in heart and testis, respectively (Fig. 2). There was a strong, broad band at higher molecular weight (5-8 kb) in spleen and testis, indicating a heterogeneous population of the transcript in these tissues. There was no significant expression in kidney, skeletal muscle, liver, brain, or lung. The low abundance of GalR3 in the brain is contrasted to the distribution of GalR1, which is significantly expressed only in brain and spinal cord (12,13), and to that of GalR2, which is expressed in both central and peripheral tissues (14,15). Given that the GalR3 cDNA was cloned from a hypothalamus library, it seems likely that there is a low level of expression of GalR3 in this part of the brain. The overall expression pattern of GalR3 suggests that the receptor may mediate galanin actions in the cardiovascular system (22,23) and in sexual behavior (24,25).
To characterize the pharmacology of the rat GalR3 receptor, the GalR3 plasmid (the 2.2-kb clone A5-3) was expressed in COS-7 cells. Binding of 125 I-porcine galanin to membranes prepared from transfected COS-7 cells demonstrated a low but reproducible level of specific binding, while binding of the radioligand to membranes from COS-7 cells transfected with  A5-3). Data shown represent the specific binding (total binding minus nonspecific binding). The counts for nonspecific binding, determined in the presence of 5 M rat galanin, were approximately 40% of the counts for total binding. The curve shows the fit of one-site hyperbolic binding by nonlinear regression analysis.

TABLE I
Ligand binding profile of the cloned rat GalR3 receptor 125 I-Porcine galanin, at 0.3 nM, was displaced by indicated peptides from membranes prepared from COS-7 cells transfected with pcDNA3.1(Ϫ)-rGalR3(A5-3) (see "Materials and Methods"), pcDNA-rGalR1 cDNA, or pCR3.1-rGalR2 cDNA (15). The expression levels for GalR1 and GalR2 were comparable (400 -500 fmol/mg) and 10 g of membrane protein were used in the assays. K i values were calculated from EC 50 values (K i ϭ EC 50 /(1 ϩ [ 125 I-ligand]/K d )) (17) determined by nonlinear regression analysis of the binding data. Data are presented as mean of K i Ϯ S.E. from two to seven independent experiments (numbers in parentheses) performed in duplicate. K i (ligand)/K i (galanin) denotes relative affinity of ligands in respect to that of native galanin.
In summary, we have cloned and characterized a novel galanin receptor subtype termed GalR3. The receptor shares homology (36 -54%) to the previously cloned GalR1 and GalR2 receptors. The distribution of GalR3 mRNA expression is strikingly different from those of GalR1 and GalR2 and appears to be restricted to the peripheral tissues. The pharmacology of the GalR3 receptor is distinguished from the other two receptors by the requirement of amino acids 17-19 of galanin (Fig. 4). The three galanin receptor subtypes show different pharmacological profiles with respect to galanin analogs, suggesting that they bind to distinct pharmacophores within the galanin peptide (Fig. 4B). This observation suggests the potential for physiological control of galanin receptor subtype activation by selective ligand modification such as differential proteolysis (27,28). The characterization of this new galanin receptor should aid in delineating specific physiological roles of the galanin receptor subtypes.