Scholarly article on topic 'Ala5-galanin (2–11) is a GAL2R specific galanin analogue'

Ala5-galanin (2–11) is a GAL2R specific galanin analogue Academic research paper on "Biological sciences"

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{Agonist / "Dynamic mass redistribution" / Galanin / "GAL2R specific ligand" / GPCR / Impedance / Neuropeptide / "Label-free real-time assay"}

Abstract of research paper on Biological sciences, author of scientific article — Kristin Webling, Johan Runesson, Andreas Lang, Indrek Saar, Barbara Kofler, et al.

Abstract It is over 30years since the regulatory peptide galanin was discovered by Professor Mutt and co-workers. Galanin exerts its effects by binding to three galanin G-protein coupled receptors, namely GAL1R, GAL2R and GAL3R. Each galanin receptor has a different distribution in the central nervous system and the peripheral nervous system as well as distinctive signaling pathways, which implicates that the receptors are involved in different biological- and pathological effects. The delineation of the galaninergic system is however difficult due to a lack of stable, specific galanin receptor ligands. Herein, a new short GAL2R specific ligand, Ala5-galanin (2–11), is presented. The galanin (2–11) modified analogue Ala5-galanin (2–11) was tested in 125I-galanin competitive binding studies for the three galanin receptors and the G-protein coupled receptor signaling properties was tested by the ability to influence second-messenger molecules like inositol phosphate and cyclic adenosine monophosphate. In addition, two different label-free real-time assays, namely EnSpire® based on an optical biosensor and xCELLigence® based on an electric biosensor, were used for evaluating the signaling properties using cell lines with different levels of receptor expression. Ala5-galanin (2–11) was subsequently found to be a full agonist for GAL2R with more than 375-fold preference for GAL2R compared to both GAL1R and GAL3R. The single amino acid substitution of serine to alanine at position 5 in the short ligand galanin (2–11) resulted in a ligand subsequently unable to bind neither GAL3R nor GAL1R, even at concentrations as high as 0.1mM.

Academic research paper on topic "Ala5-galanin (2–11) is a GAL2R specific galanin analogue"

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YNPEP-01744; No of Pages 8

Neuropeptides xxx (2016) xxx-xxx

Ala5-galanin (2-11) is a GAL2R specific galanin analogue

Kristin Webling a,*< Johan Runesson a, Andreas Lang b, Indrek Saarc, Barbara Kofler b, Ülo Langela,c

a Department ofNeurochemistry, Stockholm University, Svante Arrheniusv. 16B, SE-10691 Stockholm, Sweden

b Research Program for Receptorbiochemistry and Tumormetabolism, Laura Bassi Centre of Expertise THERAPEP, Department of Pediatrics, University Hospital Salzburg, Paracelsus Medical University, Müllner Hauptstr. 48, A-5020 Salzburg, Austria c Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia

ARTICLE INFO ABSTRACT

It is over 30 years since the regulatory peptide galanin was discovered by Professor Mutt and co-workers. Galanin exerts its effects by binding to three galanin G-protein coupled receptors, namely GAL1R, GAL2R and GAL3R. Each galanin receptor has a different distribution in the central nervous system and the peripheral nervous system as well as distinctive signaling pathways, which implicates that the receptors are involved in different biological-and pathological effects. The delineation of the galaninergic system is however difficult due to a lack of stable, specific galanin receptor ligands. Herein, a new short GAL2R specific ligand, Ala5-galanin (2-11), is presented. The galanin (2-11) modified analogue Ala5-galanin (2-11) was tested in 125I-galanin competitive binding studies for the three galanin receptors and the G-protein coupled receptor signaling properties was tested by the ability to influence second-messenger molecules like inositol phosphate and cyclic adenosine monophosphate. In addition, two different label-free real-time assays, namely EnSpire® based on an optical biosensor and xCELLigence® based on an electric biosensor, were used for evaluating the signaling properties using cell lines with different levels of receptor expression. Ala5-galanin (2-11) was subsequently found to be a full agonist for GAL2R with more than 375-fold preference for GAL2R compared to both GALiR and GAL3R. The single amino acid substitution of serine to alanine at position 5 in the short ligand galanin (2-11) resulted in a ligand subsequently unable to bind neither GAL3R nor GAI^R, even at concentrations as high as 0.1 mM.

© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Article history:

Received 27 April 2016

Received in revised form 11 August 2016

Accepted 22 August 2016

Available online xxxx

Keywords: Agonist

Dynamic mass redistribution Galanin

GAL2R specific ligand

Impedance

Neuropeptide

Label-free real-time assay

1. Introduction

It is now over 30 years since the neuropeptide galanin was discovered by Professor Mutt and colleagues (Tatemoto et al., 1983) wheniso-lating C-terminally amidated peptides from porcine intestine. Galanin was found to be widely distributed in the central nervous system (CNS) the peripheral nervous system (PNS) as well as other tissues (for review see Lang et al., 2015; Webling et al., 2012; Bauer et al., 2010; Lang and Kofler, 2011). Over the following years, attention has been drawn to this regulatory peptide due to a wide variety of physiological and pathological conditions where galanin is a part - including cancer, epilepsy, feeding, nociception and pain (for review see Lang et

Abbreviations: BBB, blood-brain barrier; CHO cells, Chinese hamster ovary cells; CNS, central nervous system; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethyloxycarbonyl; FSK, forskolin; GALjR, Galanin receptor 1; GAL2R, Galanin receptor 2; GAL3R, Galanin receptor 3; GIP, GPCR interacting proteins; GPCRs, G-protein coupled receptors; HKR, HEPES buffered Krebs-Ringer solution; HPLC, high performance liquid chromatography; IP, inositol phosphate; MBHA, p-methylbenzhydrylamine; PBS, phosphate-buffered saline; PNS, peripheral nervous system; Sar, sarcosine.

* Corresponding author.

E-mail address: kristm.webling@neurochem.su.se (K. Webling).

al., 2015; Webling et al., 2012). Galanin exerts its biological effects by binding to three G-protein coupled receptors (GPCRs), namely GALi-3R. GALiR and GAL3R are mainly signaling through Gj/o (Smith et al., 1998) while GAL2R signals through several G proteins and subsequently influence multiple intracellular signaling pathways (for review see Lang et al., 2015). The specific distribution of galanin receptor subtypes in the brain as well as different binding affinities for galanin fragments indicates different biological effects (O'Donnell et al., 1999; Smith et al., 1998). One major limitation to delineate the galaninergic system is the lack of stable, receptor subtype specific ligands. Liu et al. (2001) presented the first selective galanin ligand, a truncated peptide consisting of amino acids 2-11 (also called AR-M1896) of full-length galanin (Table 1) (Liu et al., 2001). The deletion of the first glycine resulted in loss of affinity for GAL^ (Liu et al., 2001), which has been used extensively for designing GAL2R selective ligands ever since (Runesson et al., 2009; Saar et al., 2013a, 2013b; Sollenberg et al., 2006) (Table 1). The finding that galanin (2-11) also had similar affinity to GAL3R (Lu et al., 2005a) was a step back, but galanin (2-11) is still widely used as a non-GAL1R ligand.

Over the years, reports have accumulated that the signaling cascades of GPCRs are more complex than previously anticipated. In addition to the different G protein-mediated signaling, several GPCR interacting

http://dx.doi.org/10.1016/j.npep.2016.08.008

0143-4179/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Table 1

The amino acid sequences of peptide ligands discussed.

Name Sequence Reference

Rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-amide Vrontakis etal. (1987)

Human galanin GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS Schmidt et al. (1991)

Tuna galanin GWTLNAAGYLLGPHGIDGHRTLGDKPGLA-amide Habu etal. (1994)

Human GALP APAHPGRGGWTLNSAGYLLGPVLHLPQMGDQDG Ohtaki et al. (1999)

KRETALEILDLWKAIDGLPYSHPPQPS

Galanin (2-11) WTLNSAGYLL-amide Liu et al. (2001)

AR-M1896

Human spexin NWTPQAMLYLKGAQ-amide Mirabeau etal. (2007)

Gal-B2 (Sar)WTLNSAGYLLGPKKKpalmitoyl K-amide Bulaj et al. (2008)

NAX 5055

M1145 RGRGNWTLNSAGYLLGPVLPPPALALA-amide Runesson etal. (2009)

M1153 RGRGNWTLNSAGYLLGPK(e-NH-C(O)X)-amide Saar etal. (2011)

M1160 RGRGNWLNSAGYLLGPVLPPPALALA-amide Saar etal. (2013b)

NAX 409-9 WTLNSAGYLLGPKKKKK-NH2 Metcalf et al. (2015)

Ala5-galanin (2-11) WTLNAAGYLL-amide

Sar = sarcosine; X = —CH(NH2)-CH2-CH2-COOH.

proteins (GIP) have been described (Xiao et al., 2010) (for review see Jacobson, 2015; Shukla et al., 2014; Khoury et al., 2014; Luttrell et al., 2015; Walther and Ferguson, 2015). Activated (3-arrestins have been shown to trigger a second wave of cell signaling (Ferrie et al., 2013) alongside a third wave of GPCR signaling from internalized receptors in endosomes (Irannejad et al., 2013; Irannejad and von Zastrow, 2014; Tsvetanova et al., 2015), which adds to the complexity of GPCR signaling.

These findings highlight that measuring a single second messenger to evaluate the signaling property for GPCRs that signal through multiple pathways as inadequate to show the true biological response (for review see Hall and Lefkowitz, 2002; Kenakin, 2009; Zhang and Xie, 2012).

However, to evaluate second-messengers from all known signaling pathways of certain GPCRs would be time-consuming and difficult due to biased ligands.

Since GPCR activation leads to cytoskeletal-guided transport of molecules and subsequent changes in cell adhesion and morphological re-arrangements, new real-time label free techniques have been developed to evaluate the sum of cellular events of activated GPCRs (for review see Grundmann and Kostenis, 2015; Ke et al., 2015; Lee, 2009; Nayler et al., 2010; Peters and Scott, 2009; Scott and Peters, 2010; Zhang and Xie, 2012). The sum of these cellular events gives a combined biological response measured in real time by these label-free techniques as either changes in wavelength due to mass redistribution of the cells (optical biosensor) or measuring changes in impedance due to cell adhesion changes on electrodes (an electric biosensor).

In this study, two real-time label free techniques, one based on optical techniques and one based on impedance were used alongside classical signaling measurements, i.e. cyclic adenosine monophosphate (cAMP) measurements and inositol phosphate (IP) turnover, to evaluate the signaling property of ligands and receptors.

Here we present a new short galanin receptor 2 subtype specific li-gand, Ala5-galanin (2-11), with no detectable binding to neither human GAL-iR nor human GAL3R (for sequence see Table 1). Two classical signaling methods, IP turnover and cAMP assay, are also compared to two different real-time label-free assays for GPCR signaling, for both Ala5-galanin (2-11) and full-length galanin.

2. Materials and methods

2.1. Peptide design

Galanin (1-15) is highly conserved among species (for review see Langel and Bartfai, 1998) which implicates the N-terminus to be impor-

tant for receptor binding. The removal of the first glycine along with

omission of galanin 12-29/30 of galanin has been shown to produce a non-GAL^-ligand (Liu et al., 2001; Lu et al., 2005b).

The design of Ala5-galanin (2-11), with the amino acid sequence WTLNAAGYLL-amide (see Table 1), was based on the short galanin fragment galanin (2-11) but with a single amino acid substitution where serine at position five was changed to alanine. Alanine instead of serine at position six in full-length galanin is found in yellowfin tuna (Habu et al., 1994).

2.2. Peptide synthesis

The peptides were synthesized in a stepwise manner using small scale (0.1 mmol) 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy on an automated Syro multipeptide synthesizer (MultiSynTech, GmbH, Witten, Germany) using p-methylbenzhydrylamine (MBHA) resin (Iris Biotech GmbH, Marktredwitz, Germany), which generated a C-terminally amidated peptide after final cleavage. Fmoc amino acids (Iris Biotech GmbH, Marktredwitz, Germany) were coupled as hydroxybenzotriazole (HOBt) esters. The peptides were cleaved from the resin using a solution of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) and 2.5% H2O for 3 h. The peptides were subsequently purified by reversephase HPLC (Nucleosil 120-3 C-18 column), freeze-dried and the correct molecular weight was determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (Voyager-DE STR, Applied Biosystems, Foster City, CA, USA). The freeze-dried peptides were stored at — 20 °C until used.

23. Cell culture

Bowes human melanoma cells (American type Culture Collection CRL-9607) stably expressing human GALiR (PMID: 7524088) were cultured in Eagle's minimum essential medium with GlutaMAX-1 supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% non-essential amino acids, 100 U ml-1 penicillin and 100 |ag ml-1 streptomycin.

Chinese Hamster Ovary (CHO) K1 cells stably expressing human GAL2R (a kind gift from Kathryn A. Jones and Tiina P. Iismaa, Sydney, Australia) were cultured in Dulbecco's modified essential medium F12 with GlutaMAX-1 supplemented with 10% FBS, 100 U ml-1 penicillin and 100 |ag ml-1 streptomycin.

SH-SY5Y cells stably transfected with an tetracycline inducible overexpression (see Table 2) of either GALiR or GAL2R (PMID: 14592962) were cultivated in Dulbecco's modified eagle medium supplemented with 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 100 U ml-1 penicillin and 100 |ag ml-1 streptomycin.

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Table 2

The cell lines used in this study as well as the concentration of the receptors expressed.

Cell line and receptor expressed

Concentration membrane bound receptor in

pmol/mg protein References

Bowes melanoma cells

expressing human GAL1R Chinese hamster ovary cells expressing human GAL2R Flp-In T-Rex 293 cells

expressing human GAL3R SHSY5Y stably expressing

human GAL1R SHSY5Y with tetracycline induced human GAL1R overexpression SHSY5Y stably expressing

human GAL2R SHSY5Y with tetracycline induced human GAL2R overexpression

0.54 ±0.185 0.675 ± 0.152 0.696 ± 0.293 0.4 ± 0.1 10.2 ± 1.2

0.05 ±0.016 3.2 ± 0.3

Runesson et al. (2009) Runesson et al. (2009) Runesson et al. (2009) Berger et al. (2004) Berger et al. (2004)

Berger et al. (2004) Berger et al. (2004)

Flp-InT-REx 293 GAL3Rcell line (Runesson et al., 2010) was cultured in Dulbecco's modified essential medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U ml-1 penicillin, 100 pg ml-1 streptomycin, 15 pg ml-1 blasticidin S and 150 pg ml-1 Hygromycin B (Roche Diagnostics). Tetracycline (1 pg/ml) (Sigma Aldrich, St. Louis, MO, USA) to induce the expression of GAL3R were added 24 h prior to cell harvesting for the 125I galanin competitive study, and after 24 h of cell seeding in the real-time impedance based 96-well E-plate VIEW.

Cell cultures were grown at 37 °C in a 5% CO2 incubator. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA) and cell plastics from Corning (Corning, NY, USA).

2.4. Galanin receptor binding studies

Cells for 125I-galanin receptor displacement studies were seeded in 150 mm dishes and cultured in complete cell media for 2-3 days until approximately 90% confluent. Complete cell media were changed every other day. The GAL3R Flp-In T-REx cell line had an addition of tetracycline (1 pg/ml) 24 h prior to harvest for inducing GAL3R expression. Cells were washed thrice and scraped into phosphate-buffered saline (PBS) and centrifuged twice at 4 °C, 3000 xg for 5 min.

The pellet was resuspended in assay buffer (20 mM HEPES, 5 mM MgCl2, pH 7.4) supplemented with EDTA (5 mM EDTA) and incubated on ice for 45 min before centrifugation at 4 °C, 8500g for 15 min. After washing the pellet in assay buffer and repeated centrifugation the obtained pellet was resuspended in assay buffer supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and stored at - 20 °C. Protein concentration was determined according to Lowry (BioRad, Stockholm, Sweden). Displacement studies on cell membranes were performed in a final volume of 200 pl, containing 0.1 nM porcine-[125I]-galanin (2200 Ci/mmol, Perkin-Elmer Life Science, Boston, MA, USA), 30 pg cell membrane, and various concentrations of peptide (10-9 to 10-4 M). Peptide solutions were prepared in assay buffer supplemented with 0.3% bovine serum albumin (BSA) using silanized (dichlorodimethylsilane, Sigma-Aldrich, St. Louis, MO, USA) tubes and pipette tips to prevent unspecific binding. Samples were incubated at 37 °C for 30 min while gently shaking after which the samples were transferred and filtered through a MultiScreen-FB filter plate (Millipore, Billerica, MA, USA) pre-soaked in 0.3% polyethylenimine solution (Sigma-Aldrich, St. Louis, MO, USA) using vacuum. The filters were washed thrice with assay buffer and the retained radioactivity was determined in a (3-counter (Tri-Carb Liquid Scintillation Analyser, model 2500 TR, Packard Instrument Company, Meriden, CT, USA) using OptiPhase Supermix Cocktail (Perkin-Elmer Life Science, Boston, MA, USA) as scintillation fluid. IC50 values for the peptides were calculated using Prism 6.0 (GraphPAD Software Inc., San Diego, CA, USA)

and converted into Ki values using the equation of Cheng-Prusoff (Cheng and Prusoff, 1973).

2.5. Cyclic adenosine-monophosphate (cAMP) measurements

SH-SY5Y cells stably expressing either GAL^ or GAL2R were seeded into 48-well plates and grown to confluency. Tetracycline was not added since the experiment aimed to examine a lower receptor expression level (see Table 2). Cells were incubated for 2 h with DMEM and [3H]adenine (3 pCi/ml) (Perkin Elmer, Waltham, MA, USA) and then washed thrice with Hank's balanced salt solution (Gibco) before stimulated with 20 pM forskolin and/or peptide ligand Ala5-galanin (2-11) (10-5 to 10-7) for 30 min at 37 °C in the presence of inhibitors (1 mM 3-isobutylmethylxanthine (IMBX) (Sigma, Vienna, Austria), 1 pM BAY 60-7550 (Santa Cruz, Heidelberg, Germany) and 10 pM rolipram (Sigma, Vienna, Austria)). The cells were subsequently lysed using of 5% (w/v) trichloroacetic acid followed by the addition of 250 pl of 5% (w/v) trichloroacetic acid supplemented with 0.1 mM cAMP and 0.1 mM adenosine triphosphate (ATP) (Sigma, Vienna, Austria) for at least 20 min at 4 °C.

The lysates were then added to Dowex 50W-X4 columns (200400 mesh) (Bio-Rad, Hercules, CA, USA) and washed twice with deion-ized water before placed over alumina columns and eluted with 0.1 M imidazole (AppliChem, Darmstadt, Germany). Radioactivity of the elu-ate was determined in a liquid scintillation counter (Tri-Carb model 1600 TR; Packard Instrument Company) using Ultima Gold XR (Perkin Elmer, Waltham, MA, USA) as scintillation fluid.

2.6. Inositol phosphate accumulation

CHO K1 cells stably expressing human GAL2R were seeded in 12-well plates and cultured in complete cell culturing media for 48 h before incubation with 1 pCi [3H]-myo-inositol (Perkin Elmer) in M-199 medium (Invitrogen, Carlsbad, CA, USA) containing 100 U ml-1 penicillin and 100 pg ml-1 streptomycin for 24 h. Prior to experiments, cells were incubated with fresh M-199 medium for 10 min at 37 °C and washed twice with HEPES buffered Krebs-Ringer solution (HKR) buffer (5 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 2.05 mM MgCl2, 1.8 mM CaCl2,1 g l-1 glucose, pH 7.4) followed by 10 min pre-incubation in 800 pl HKR buffer with 10 mM LiCl at 37 °C. Cells were then stimulated with peptide in various concentrations (10-9 to 10-4 M) generating a total volume of 1000 pl. After addition of peptide the samples were incubated for 1 h at 37 °C. The reaction was terminated by addition of 200 pl ice cold 20% perchloric acid followed by incubation on ice for 30 min. The samples were neutralized to pH 7 by addition of 1.5 M KOH/75 mM HEPES. Anion exchange chromatography of the samples was performed over 1.5 cm 50:50 Dowex (AG 1-X8 Resin, 200400 mesh formate, BioRad, Hercules, CA, USA). The columns were washed with 5 ml distilled water before the inositol phosphates (IP) were eluted with 5 ml of 0.1 M formic acid/1.2 M ammonium formate. Radioactivity of the eluate was determined in a (3-counter (Tri-Carb Liquid Scintillation Analyzer, model 2500 TR, Packard Instrument Company, Meriden, CT, USA) using Utima Flo AF (Perkin-Elmer Life Science, Boston, MA, USA) as scintillation fluid. Each sample was normalized against the total count obtained before anion exchange chromatography.

2.7. Label-free assay for signaling using EnSpire®

SH-SY5Y cells stably expressing either GALiR or GAL2R were seeded in EnSpire LFC-348 well plates (Perkin Elmer, Austria) at a concentration of 12,000 cells per well and cultured overnight at 37 °C and 5% CO2. Tetracycline was not added to be able to examine the signaling in cells expressing the receptor at lower concentrations. The media was then aspired and the cells were washed four times with Hank's balanced salt solution (HBSS) buffer supplemented with HEPES (20 mM) and the

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final volume was adjusted to 30 per well. The plate was allowed to equilibrate to assay temperature for 1 h in the Enspire multimode reader (Perkin Elmer, Austria). HBSS buffer supplemented with HEPES (20 mM) alone or dilution series of Ala5-galanin (2-11) dissolved in the same buffer were prepared and placed on a compound plate which were allowed to equilibrate to reach the same temperature as the Enspire plate holding the cells. A baseline reading was first generated by 4 measurements before ligands were added. The plate was measured between 30 and 70 repeats and each experiment was performed in triplicates. The mean was used to generate the dose response curve and EC50 values were calculated using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).

2.8. Label-free assay for signaling using xCELLigence

One day prior to the experiment, 100 |al of complete cell culture media was added to each well and background recorded. Following background measurement, 100 |al of complete cell media containing the cell suspension were seeded on the 96-well E-plate VIEW™ (ACEA, San Diego, USA), incubated at room temperature for 30 min and then placed onto the device station (Roche Applied Science and ACEA Biosciences) hosted inside an incubator at 37 °C with 5% CO2. The optimal cell density for the assay was determined to be 40,000 cells per well for Bowes human melanoma cells stably expressing GAL1R, 25,000 cells per well for CHO K1 cells stably expressing GAL2R and 20,000 cells per well for Flp-In T-REx 293 cells with inducible GAL3R expression. The cells were allowed to equilibrate for 24 h (48 h for the GAL3R cell line where tetracycline (1 ^g/ml) was added 24 h after seeding to induce the GAL3R expression) and impedance was constantly monitored every minute throughout the whole experiment. Prior to galanin ligand addition, complete cell media were replaced with 180 low-FBS (1%) containing cell media, according to the manufacturer's instructions, and incubated for 1 h. The ligands were dissolved in PBS at concentrations 10x the wanted final concentration and 20 were added gently to each well.

Data were analyzed using the integrated software package expressing changes in cell electrode impedance as changes in cellular index, CI, (Yu et al., 2006) and normalized to the cell index at the time of ligand addition. Concentration effect curves were generated by calculation of the area under curve (AUC) between 0 and 3600 s after peptide addition, and plotted against ligand concentration. The subsequent calculation of EC50 values was performed by non-linear regression using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).

2.9. Data analysis

For all experiments are data reported as mean ± SEM of at least three individual experiments performed in at least duplicates. Statistical significance were graded after *p-value < 0.05; **p-value < 0.01 and ***p-value < 0.001 using ANOVA Bonferroni's multiple comparison test using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Galanin receptor binding studies

al., 2012) allows full-length galanin from either of these three species to be used as controls.

The displacement of both full-length rat galanin as well as galanin (2-11) revealed for galanin a K of 1.70 ± 1.2 nM for GAL1R, 2.71 ± 0.9 nM for GAL2R and 4.33 ± 0.6 nM for GAL3R. Galanin (2-11) had no detectable binding to GAL1R, and the K for GAL2R was 14.6 ± 2.2 nM and for GAL3R 186 ± 74 nM (Fig. 1, Table 3). Ala5-galanin (211) had a lower affinity for GAL2R compared to galanin (2-11) with a Ki of 258 ± 68 and 16.6 ± 3.5, respectively, but Ala5-galanin (2-11) had no detectable binding to neither GAL1R nor GAL3R even at doses as high as 0.1 mM (Table 3, Fig. 1). In Table 3, the highest experimentally tested concentration is presented when no Ki could be calculated and indicated by a > meaning that the true K value would be higher. Consequently, Ala5-galanin (2-11) has more than 375-fold preference for GAL2R over both GAL1R and GAL3R.

3.2. Cyclic adenosine-monophosphate stimulation assay

The ability of Ala5-galanin (2-11) to initiate GPCR signaling were examined by its ability to change the concentration of cyclic adenosine-monophosphate, cAMP, in SH-SY5Y cells expressing GAL2R in a 10fold lower concentration than the CHO K1 cells. Signaling through Gi/o would decrease the cAMP concentration and forskolin was therefore used to stimulate cAMP production of the cells. Three different ligand concentrations (0.1, 1 and 10 ^M) were tested together with 20 |jM forskolin, using forskolin (20 |jM) together with 0.1 |jM full-length human galanin as control. No significant change in cAMP concentration after addition of Ala5-galanin (2-11) could be seen even at 10 |jM concentration using ANOVA Bonferroni's multiple comparison test whereas forskolin together with full-length galanin caused a statistical significant increase in cAMP concentration (**p-value < 0.01) (Fig. 2).

3.3. Inositol phosphate accumulation

The ability of Ala5-galanin (2-11) and full-length rat galanin to induce GPCR signaling was examined by the ability of the peptides to stimulate inositol phosphate (IP) production in CHO cells stably expressing human GAL2R. The ability to induce maximum IP accumulation is similar to that of full-length rat galanin and also the previously published full agonist M1145 (Runesson et al., 2009). Ala5-galanin (2-11) has relatively low affinity compared with the previously published GAL2R selective ligands with an EC50 value of 1.01 ±0.18 |jM but considered a full agonist (Fig. 3) (Runesson et al., 2009; Saar et al., 2013a, 2013b; Sollenberg et al., 2006). The EC50 value of full-length rat galanin in the present study was 66.4 ± 13 nM for GAL2R, a value in concordance with previous studies were full-length rat galanin had a EC50-value of 75 nM (Runesson et al., 2009). Using ANOVA Bonferroni's multiple comparison significance were graded according *p-value < 0.05; **p-value < 0.01 and ***p-value < 0.001. Galanin showed significant accumulation of inositol phosphate above 0.1 ^M (***p-value) whereas Ala5-galanin (2-11) had (*p-value) accumulation of inositol phosphate at 0.1 |aM and significant accumulation above 1uM (***p-value).

No significant change in IP production could be seen when Ala5-galanin(2-11) was added to Bowes melanoma cells stably expressing GAL1R nor Flp-In T-REx 293 with inducible expression of GAL3R (data not shown).

125I-galanin-receptor displacement studies were performed using full-length rat galanin, galanin (2-11) and Ala5-galanin (2-11) on membrane preparations of Bowe's human melanoma cells stably expressing human GAL-^, CHO K1 cells stably expressing human GAL2R and Flp-In T-REx 293 with inducible expression of human GAL3R. The high degree of sequence homology between rat and human galanin receptors and the reported similar binding affinity for full-length rat, human and porcine galanin to those two receptors (Wang et al., 1997; Borowsky et al., 1998; Lu et al., 2005a, 2005b; reviewed in Webling et

3.4. Label free methods using EnSpire® and xCELLigence®

When using the optical based label-free real-time system, Enspire, the ligand Ala5-galanin (2-11) was only able to generate a dynamic mass redistribution (DMR) response in the SH-SY5Y cells stably expressing GAL2R with an EC50-value of 3.23 ± 0.4 ^M. Full-length human galanin was used as control and had in this study an EC50-value of 1.08 ± 3.19 nM at SH-SY5Y cells expressing GAL1R and 47.2 ± 5.3 nM for SH-SY5Y cells expressing GAL2R.

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Fig. 1. Displacement studies of porrine-[125I]-galamn on CHO K1 cells stably expressing human GAL2R presented in open squares, and Flp-In T-REx 293 cells with inducible expression of human GAL3R presented in filled triangles, using the non-GAL1R ligand galanin (2-11) (A) and the specific GAL2RligandAla5-galanin (2-11) (B). Data is presented as mean ± SEM of three individual experiments performed in duplicates.

When using the impedance based label-free real-time assay, xCELLigence, Ala5-galanin (2-11) was only able to generate a dose-response curve in CHO K1 cells stably expressing GAL2R. The EC50-value was 191 ±49 nM (Table 4). No response could be observed when Ala5-galanin (2-11) was added to Bowes Melanoma Cells stably expressing GAL^ nor Flp-In T-REx 293 cells with tetracycline induced expression of GAL3R (data not shown). Full-length rat galanin were used as a control and had in this study EC50-values of 1.1 ± 0.11 nM for GAL^, 8.26 ± 1.9 nM for GAL2R and 412 ± 38 nM for GAL3R.

4. Discussion

The usage of knockout (KO)-animals, overexpression (OE)-animals and the use of a few available selective ligands for the different galanin receptor subtypes indicate specific involvement of galanin receptor subtypes in different diseases (for review see Webling et al., 2012). GAL2R agonists are for example considered to be neuroprotective in both Alzheimer's disease (Pirondi et al., 2010) and in kainic acid induced cell death (Webling et al., 2015), antidepressant and anxiolytic (Kuteeva et al., 2008), and able to reduce the severity of seizures in epilepsy (Robertson et al., 2010; Mazarati et al., 2004) (for review see Webling et al., 2012). This has generated an increasing interest to generate and use specific ligands to delineate the effects of the galanin receptors, but the pharmacological tools available are still limited. The first specific galanin ligand presented was galanin (2-11) (Liu et al., 2001) and even though the finding that galanin (2-11) binds with similar affinity to both GAL2R and GAL3R (Lu et al., 2005a, 2005b), it is commonly used as an non-GAL1R ligand.

Even though the N-terminal amino acid sequence ofgalanin is highly conserved among species and considered important for receptor binding, some N-terminal alterations have been shown to retain receptor binding affinity. Bulaj and colleagues reported a N-terminal methylated galanin ligand where the first glycine was replaced by sarcosine along

Table 3

Experimental Ki determined by displacement studies of porcine-[125I]-galanin on Bowes Melanoma Cells expressing human GAL1R, CHO K1 cells stably transfected with human GAL2R or Flp-In T-REx 293 cells with inducible expression of human GAL3R. The highest concentration used in this study served as a Ki-value when no experimentally Ki-value could be determined. Data are presented as mean values ± SEM of at least three individual experiments each performed in duplicates. When Ki-values could not be determined was the Ki presented as above the highest concentration experimentally used.

Name Ki (nM)

GAL,R gal2r GAL3R

Rat galanin 1.70 ± 1.2 2.71 ± 0.9 4.33 ± 0.6

Galanin (2-11) > 100,000 14.6 ± 2.2 186 ± 74

Ala5-galanin (2-11) > 100,000 258 ± 68 > 100,000

with cationic amino acids C-terminally together with a fatty acid to improve stability (Bulaj et al., 2008). The presented ligand, named NAX 5055, had nM affinity for both GALiR and GAL2R with a 14-fold preference for GAL1R over GAL2R. Unfortunately, no data for NAX 5055 and GAL3R affinity have been reported to date. The first GAL2R specific ligand M1145 presented by Runesson et al. (2009) was designed with a four amino acid sequence from the GAL2R/GAL3R selective galanin-like peptide (GALP) added to the N-terminus to improve the GAL2R selectivity alongside replacement of the first glycine to asparagine to minimize binding to GAL1R (Runesson et al., 2009) (Table 1). M1145 retained a nM binding affinity for GAL2R and had a 90-fold preference for GAL2R over GAL1R and a 76-fold preference for GAL2R over GAL3R (Runesson et al., 2009). Based on the M1145 sequence, a new ligand with improved specificity for GAL2R was designed and presented by Saar et al., 2011,

Fig. 2. Signal transduction using cyclic adenosine-monophosphate stimulation. Cyclic adenosine-monophosphate, cAMP, production was measured radiometrically. Ala5-galanin (2-11) did not significantly stimulate cAMP production in SH-SY5Y cells stably expressing human GAL2R Full-length human galanin (0.1 |jM) together with forskolin (FSK) were used as control. Data shown are mean values of triplicates ± SEM of one experiment. **p-Value < 0.01, ANOVA Bonferroni's multiple comparison test.

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il 400H

c a ® ,5

£ ® 0- 200-

> rat galanin 1-29 . Ala5 -galanin (2-11)

-12 -10 -8 -6 -4 -2

log[peptide], M

Fig. 3. Signal transduction via inositol phosphate accumulation. CHO K1 cells stably expression human GAL2R were pre-incubated with [3H]-myo-inositol for 24 h previous to galanin ligand addition at different concentrations (10-10-10-4 M). The dose response curve of Ala5-galanin (2-11) is plotted using filled squares and full-length rat galanin plotted as filled circles. Data are presented as % of control when no ligand was added as mean ± SEM of six individual experiments each performed in duplicates.

named M1153 (Table 1) (Saar et al., 2011). The C-terminal branching produced a ligand that retained nM affinity for GAL2R with a 380-fold preference for GAL2R over GAL^ and a 46-fold preference for GAL2R over GAL3R. The seventh amino acid in the M1145 sequence, threonine, was deleted to reduce the affinity for GAL3R according to mutagenesis studies resulting in an even more selective GAL2R specific ligand, named M1160 (for sequences see Table 1) (Saar etal., 2013b). M1160 had a 465-fold preference for GAL2R over GALiR and more than a 1000-fold preference for GAL2R over GAL3R. In Ala5-galanin (2-11), the substitution of serine to an alanine at position five in galanin (211) reduced one hydroxyl group in the peptide. The re-evaluation of an alanine scan to identify important pharmacophores for GAL2R showed a 6-fold reduction in EC50-value while replacing the serine at position five in galanin (2-11) (Lundström et al., 2005). Land et al. (1991) showed a similar 6-fold reduction in KD while comparing galanin (1-16) to Ala6-galanin (1-16) (Land etal., 1991). In an in silico study using the short galanin fragment, galanin 2-6, for docking to GAL3R highlighted serine as important for forming a hydrogen bond to Ala92322 (Runesson et al., 2010). In concordance with the experimental result presented herein, where the importance of serine at position five were highlighted by replacing serine with alanine resulted in a ligand unable to bind to GAL3R even at high concentrations of 0.1 mM.

The C-terminus of galanin has been reported as an important sequence for binding to the GAL3R even though the GAL3R binding pocket has been described as tighter and more narrow as compared to GAL1R and GAL2R (Runesson et al., 2010). Our new GAL2R specific ligand, Ala5-galanin (2-11), behaves as a full agonist for GAL2R when examined by 125I-galanin competitive binding studies as well as IP turnover studies (Table 3; Figs. 1 and 2). Ala5-galanin (2-11) has a lower affinity for the receptor compared to other published GAL2R selective ligands, but is the shortest peptide fragment that shows excellent receptor specificity with no detectable binding to neither GAL^ nor GAL3R even at doses as high as 0.1 mM. In concordance with previously published specific GAL2R agonists (M1145 (Runesson et al., 2009), M1153 (Saar et al.,

Table 4

Galanin receptor signaling studies using a real-time label-free technique based on impedance - the xCELLigence system.

EC50 values obtained from the impedance based (RT-CES) xCELLigence system using Bowes Melanoma Cells stably expressing GAL1R, CHO K1 cells stably expressing GAL2R and Flp-In T-REX 293 cells with tetracycline induced expression of GAL3R. Dose-response curves were generated and calculated by area-under-curve between 0 and 3600 s after peptide addition, and plotted against the ligand concentration. Data from at least three representative experiments performed in duplicates, are presented as mean ± SEM.

EC50 (nM)

Name GAL1R gal2r GAL3R

Rat galanin 1 -29 1.1 ± 0.11 8.26 ± 1.9 412 ± 38

Ala5-galanin (2-11) >100,000 191 ±49 >100,000

2011) and M1160 (Saar et al., 2013b)) is the binding affinity for GAL2R lower than for full-length galanin and galanin (1-16). Our new GAL2R specific ligand, Ala5-galanin (2-11), could be used as a research tool to delineate the galaninergic system and the role of GAL2R in several studies where galanin (2-11) has been used to exclude effects caused by interactions with GAL3R.

Limitations for this ligand, among most peptides, are the fast degradation when used in animal studies along with problematic administration routes were i.c.v injections are often used since unmodified peptide ligands are unable to pass the blood-brain barrier (BBB). Bulaj and co-workers presented a new modification that increased the half-life of galanin analogues from minutes to several hours by adding several positive amino acids along with a fatty acid at the C-terminus (Bulaj et al., 2008; Robertson et al., 2010). A study by Saar et al. (2013a) have adopted the modifications from Bulaj and co-workers to produce stable GAL2R selective ligands that could pass the BBB when administrated intravenously and exert anti-depressant affects in mice (Saar et al., 2013a). Recent studies by White and colleagues presents a C-terminally monodisperse oligoethyleneglycol (dPEG) modified GAL2R selective galanin ligand, named NAX 409-9, that is stable and peripherally active with restricted BBB penetration (Zhang et al., 2013; Metcalf et al., 2015).

Recently, a 14 amino acid long peptide named spexin was reported as a GAL2/3R ligand with a higher potency for GAL3R than galanin (for sequence see Table 1) (Kim et al., 2014). Human spexin is C-terminally amidated like galanin from most species (human and macaque being exceptions) and share Trp2, Thr3, Tyr9, Leu10 and Gly12 (Mirabeau et al., 2007; Schmidt et al., 1991 ; Cunningham et al., 2002). Based on the spexin sequence, Reyes-Alcaraz et al. (2016) reported several new GAL2R specific ligands when replacing amino acids in the spexin sequence by the corresponding amino acid from galanin and highlighted the importance of Gln5, Met7, Lys11 and Ala13 in spexin for GAL3R binding (Reyes-Alcaraz et al., 2016). The presented GAL2R specific ligands, Asn5-spexin, Ala7-spexin, Phe11-spexin, Pro13-spexin and Asn5, Ala7, Phe11, Pro13-spexin, all retained similar binding affinity to GAL2R as spexin but no binding to neither GAL^ nor GAL3R could be detected even at concentrations of 10 ^M (Reyes-Alcaraz et al., 2016). Additionally, the spexin based GAL2R specific ligands were chemically stabilized by either addition of Fmoc, N-terminal PEGylation, C-terminal or N-ter-minal substitution of D-amino acids which resulted in an up to four times improved serum stability compared to spexin (Reyes-Alcaraz et al., 2016).

The signaling cascade of GPCRs has been shown to involve both G protein-dependent and -independent pathways. For GAL2R, it has been shown that the Ga subtypes used upon activation are mainly Gq/11, but Gi/o, and G12/13, are also involved (for review see Lang et al., 2015; Wittau et al., 2000). Thus to get a summarized picture using classical signaling assays is very time consuming. Signaling through Gq causes an increase in IP production whereas signaling through Gi/o is classically accompanied by decreasing cAMP levels. The contribution of G12/13 is more difficult to access using classical assays (Wettschureck, 2005).

In the IP turnover study classically measuring Gq signaling, Ala5-galanin (2-11) behaved as a full agonist compared to full-length rat galanin on CHO K1 cells overexpressing GAL2R in agreement that Gq is the main signaling pathway for GAL2R. By comparing the classical signaling assays with the new real time label free techniques, full-length galanin showed similar values to GAL^ and GAL2R, with 1.1 ± 0.11 nM and 8.26 ± 1.9 nM, respectively, while the EC50 towards GAL3R was 412 ± 38 nM. The xCELLigence system has similar or improved sensitivity when compared to traditional endpoint assays performed in the same laboratory. The relative low EC50 for full-length galanin at GAL3R could reflect intrinsic receptor properties or might be related to the utilized cell clone. Runesson et al. (2009) reported an EC50-value of 530 nM for galanin on GAL3R using a GTP^-assay and the receptor expression of GAL3R is similar to those of GALiR and GAL2R used in this study (Runesson et al., 2009).

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In this study, the lack of statistically relevant results by Ala5-galanin (2-11) in the cAMP stimulation assay using cells expressing GAL2R at a 10-fold lower concentration compared to the CHO K1 cell line could be due to Ala5-galanin (2-11) being a biased ligand i.e. a ligand that prefers one signaling pathway over several others and the lower receptor expression might be difficult to evaluate using this assay. Signaling through Gi/o will decrease cAMP levels and therefore forskolin was added to stimulate the cells. However, full-length galanin caused a significant increase in cAMP. Increased cAMP levels are classically related to activation through Gs, but GAL2R has not been reported to signal through this G-protein (Lang et al., 2015; Lang et al., 2007; Wittau et al., 2000). However, Gu et al., (1994) reported that full-length galanin added to dispersed smooth muscle cells from guinea pig stomach caused increased levels of cAMP (Gu et al., 1994). Signaling through Gq activates protein kinase C (PKC) and causes an increase in intracellular Ca2+ concentration via IP (Lang et al., 2015), both PKC and Ca2+ have been reported to stimulate adenylyl cyclase (AC) which in turn leads to increased cAMP levels (Mons et al., 1998). This might explain the small change in cAMP concentrations for full-length galanin since both cAMP stimulating as well as cAMP decreasing routes could be activated. If comparing classical signaling alongside the new label-free real-time assays according to cell line used it is obvious that similar or somewhat better sensitivity can be observed using label-free assays. The signaling cascade can be further evaluated using the real-time label-free techniques together with inhibitors, as shown by Bouvier and colleagues that presented an additional signaling route for the ß2-adrenergic receptor (Stallaert et al., 2012). These techniques can also be used to evaluate primary cell cultures, an interesting aspect since GPCRs recently have been reported to exist as heteromers in vivo (Borroto-Escuela et al., 2014; Fuxe et al., 2012; Narvaez et al., 2015). But in this study, the real-time label-free techniques were used to evaluate their potential use as a screening method for novel galanin receptor ligands.

5. Conclusions

Ala5-galanin (2-11) was found to be the shortest GAL2R specific agonist and the importance of serine at position five from a previous in silico study of GAL3R was here experimentally verified. Two classical second messenger assays for GPCR signaling was compared with two label-free real-time techniques on both high receptor expressing cells as well as a lower receptor expressing cell lines resulting in similar or somewhat better sensitivity observed for the label-free techniques.

Acknowledgment

We would like to express our gratitude to Tiina P. Iismaa and Kathryn A.Jones (Neurobiology Program, Garvan Institute of Medical Research, Sydney, Australia) for the kind gift of CHO K1 cells stably expressing human GAL2R. Linda Lundström and Silvia Gatti-McArthur (F. Hoffmann-La Roche AG, Basel, Switzerland) for Flp-In T-REX 293 cell line with inducible expression of human GAL3R.

This work was supported by grants from Olle Engkvist Byggmästares Foundation (JR), the Swedish Research Council (VR-Med) (K2012-66X-21148-04-5) (ÜL) (521-2011-2461), and the Austrian Research Promotion Agency (822782/THERAPEP) (BK).

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