Cell-Penetrating Mimics of Agonist-Activated G-Protein

Coupled Receptors

Pernilla Ostlund,1Kalle Kilk,

2Maria Lindgren,

1Mattias Hallbrink,

1Yang Jiang,

1

Metka Budihna,3Katarina Cerne,

3Aljosa Bavec,

4Claes-Goran Ostenson,

5Matjaz Zorko,

4

and Ulo Langel1,6

(Accepted November 1, 2005)

Cell-penetrating peptides have proven themselves as valuable vectors for intracellular delivery.Relatively little is known about the frequency of cell-penetrating sequences in native proteinsand their functional role. By computational comparison of peptide sequences, we recently

predicted that intracellular loops of G-protein coupled receptors (GPCR) have highprobability for occurrence of cell-penetrating motifs. Since the loops are also receptor andG-protein interaction sites, we postulated that the short cell-penetrating peptides, derived

from GPCR, when applied extracellularly can pass the membrane and modulate G-proteinactivity similarly to parent receptor proteins. Two model systems were analyzed as proofs ofthe principle. A peptide based on the C-terminal intracellular sequence of the rat angiotensin

receptor (AT1AR) is shown to internalize into live cells and elicit blood vessel contractioneven in the presence of AT1AR antagonist Sar1-Thr8-angiotensin II. The peptide interactswith the same selectivity towards G-protein subtypes as agonist-activated AT1AR andblockade of phospholipase C abolishes its effect. Another cell-penetrating peptide, G53-2

derived from human glucagon-like peptide receptor (GLP-1R) is shown to induce insulinrelease from isolated pancreatic islets. The mechanism was again found to be shared with theoriginal GLP-1R, namely G11-mediated inositol 1,4,5-triphosphate release pathway. These

data reveal a novel possibility to mimic the effects of signalling transmembrane proteins byapplication of shorter peptide fragments.

KEY WORDS: Angiotensin; cell-penetrating peptide; glucagon-like peptide 1; G-protein coupledreceptors; signal transduction.

INTRODUCTION

Several peptides, called cell-penetrating peptides(CPPs), have been shown to internalize into livingcells with high efficiency (see Zorko and Langel(2005) for review). The actual mechanism for entry ofthese peptides is an ongoing subject of intenseresearch. CPPs can be divided into two classes basedon their origin, protein derived and synthetic/chime-rical peptides. So far, CPPs have been successfullyused for transport of hydrophilic molecules into cells,which normally are not internalized, such as proteins

1 Department of Neurochemistry and Neurotoxicology, Stockholm

University, S-106 91 Stockholm, Sweden.2 Centre of Molecular and Clinical Medicine, University of Tartu,

Ravila 19, 51014 Tartu, Estonia.3 Institute of Pharmacology and Experimental Toxicology, Medi-

cal Faculty, University of Ljubljana, Korytkova 2, 1000 Ljublj-

ana, Slovenia.4 Institute of Biochemistry, Medical Faculty, University of

Ljubljana, Lipiceva 2, 1000 Ljubljana, Slovenia.5 Department of Molecular Medicine, Endocrine and Diabetes

Unit, Karolinska Hospital, S-171 76 Stockholm, Sweden.6 Correspondence should be addressed to: Ulo Langel, Department

of Neurochemistry and Neurotoxicology, Stockholm University,

S-106 91 Stockholm, Sweden. Tel: +46-8-164368; Fax +46-8-

161371; e-mail: ulo@neurochem.su.se.

International Journal of Peptide Research and Therapeutics, Vol. 11, No. 4, December 2005 (� 2005), pp. 237–247

DOI: 10.1007/s10989-005-9329-9

2371573-3149/05/1200–0237/0 � 2005 Springer Science+Business Media, Inc.

and PNA oligomers (see Dietz and Bahr (2004) forreview).

Here we demonstrate that two transmembraneproteins can serve as a source for production of novel,functionally effective CPPs. Angiotensin receptors aremembers of the 7-transmembrane G-protein coupledreceptor (GPCR) super family and are importantcomponents of the blood pressure and electrolytebalance maintaining system in mammals. They existin two types: AT1R, where two subtypes have beenidentified in rodents (AT1AR and AT1BR), andAT2R, among which AT1R seems to be responsiblefor the mediation of almost all known systemic effectsof angiotensin II (Inagami 1999; Brede and Hein2001). AT1R is involved in contraction of smoothmuscles in different tissue, e.g. in blood vessels,uterus, bladder, and some endocrine glands, and thereceptors are also distributed widely in kidney, liver,and in the CNS (Brede and Hein 2001). Signalingthrough AT1Rs is mediated via inhibition of adenylylcyclase (activation of Gi/Go) and via modulation ofphosphoinositide metabolism, most probablythrough the pertussis toxin-insensitive G-proteins Gq

and G11 (Touyz and Berry 2002). Antagonists ofAT1R are potential antihypertensive drugs and somenon-peptide antagonists, e.g. lorsatan, have beensuccessfully introduced into clinical use (Goodfriendet al. 1996). Selective agonists for AT1R, however,are not available today. Agonists would be of interestas potential drugs for vasoconstriction, e.g. chronicalhypotension or migraine.

The glucagon-like peptide-1 receptor (GLP-1R) isanother member of the 7-transmembrane spanningGPCR super family. The receptor is expressed inpancreatic b-cells, brain (Shimizu et al. 1987; Utten-thal et al. 1992) and mRNA expression has beenfound in lung, brain, kidney, stomach and heart (Weiand Mojsov 1995). GLP-1R is activated by theincretin hormone GLP-1 and is responsible for thepotentiation of glucose-stimulated insulin release.The signaling pathways involved in GLP-1 inducedinsulin secretion include signaling through adenylylcyclase (Goke and Conlon 1988; Goke et al. 1989; Luet al. 1993; Widmann et al. 1994), phospholipase C(Dillon et al. 1993; Wheeler et al. 1993; Galera et al.1996; Marquez et al. 1998) and phosphatidyl inositol3-kinase (reviewed in MacDonald et al. (2002)).Accordingly, studies show that the GLP-1R couplesto Gs and Gq/11 (Montrose-Rafizadeh et al. 1999;Hallbrink et al. 2001). Point mutations of the recep-tor reveal that the third intracellular loop is impor-tant for signaling (Heller et al. 1996; Takhar et al.

1996; Mathi et al. 1997). Since GLP-1 induces insulinrelease it has been proposed as a therapeutic fornon-insulin-dependent diabetes mellitus (NIDDM),together with several GLP-1 analogs, e.g. exendin-4and liraglutide which are resistant to proteolyticcleavage, which are under development (Baggio andDrucker, 2004).

Synthetic peptides, derived from the intracellularloops of GPCRs, can influence receptor–G-proteininteractions with the original receptor in membranepreparations (Bommakanti et al. 1995; Shirai et al.1995; Rezaei et al. 2000; Hallbrink et al. 2001; Bavecet al. 2003). For the AT1AR, peptides correspondingto fragments of the C-terminal tail (Shirai et al. 1995;Franzoni et al. 1997), the third (Conchon et al. 1997)and even the second (Thompson et al. 1998) intra-cellular loops are by this way found to affect signaltransduction. For the GLP-1R, the third intracellularloop seems to be involved in transmitting the signal,whereas the first and second intracellular loopsexhibit modulatory roles (Hallbrink et al. 2001; Ba-vec et al. 2003). To allow entry of the peptides de-rived from GPCRs into intact cells, they have beencoupled to CPPs (Howl et al. 2003). In the currentwork, we present an all-in-one approach, where thereceptor-derived peptide is, in itself, a CPP. We havesynthesized the peptide M511, derived from the C-terminal intracellular part of the rat AT1AR, and thepeptides G53-1 and G53-2, derived from the thirdintracellular loop of the rat GLP-1R. All three pep-tides internalize into live cells and M511 and G53-2display properties mimicking the effects of theirrespective parent protein.

MATERIALS AND METHODS

Peptide Synthesis

Peptides were synthesized in a stepwise manner in a 0.1 mmol

scale on a peptide synthesizer (Applied Biosystems model 431A,

USA) using t-Boc strategy of solid-phase peptide synthesis. tert-

Butyloxycarbonyl amino acids (Bachem, Bubendorf, Switzerland)

were coupled as hydroxybenzotriazole (HOBt) esters to a p-meth-

ylbenzylhydrylamine (MBHA) resin (Bachem, Bubendorf, Swit-

zerland) to obtain C-terminally amidated peptide. 5(6)-

Carboxyfluorescein was coupled manually to the N-terminus by

adding a 3-fold excess of HOBt and N, N¢-diisopropylcarbodiimide

(DIC)-activated biotin (Chemicon, Stockholm, Sweden) in DMF to

the peptidyl-resin. The peptide was finally cleaved from the resin

with liquid HF at 0�C for 45 min in the presence of p-cresol. The

purity of the peptide was >98% as demonstrated by HPLC on an

analytical Nucleosil 120-3 C-18 RP-HPLC column (0.4�10 cm)

and the correct molecular mass was obtained by using a plasma

238 Ostlund et al.

desorption mass spectrometer (Bioion 20, Applied Biosystems,

USA).

Cell Culture

All cell culture reagents were from Invitrogen, Sweden. The

mouse neuroblastoma cell line (N2a) (ATCC via LGC, Sweden)

was propagated in Dulbecco’s Modified Essential Media (DMEM)

with Glutamax supplemented with 10% fetal bovine serum, 1 mM

sodium pyruvate, non-essential amino acids 1�100, 100 U/ml

pencillin and 100 lg/ml streptomycin (all from Invitrogen, Swe-

den). Rat insulinoma RINm5F cells were cultivated in RPMI with

Glutamax supplemented with 10% fetal bovine serum, 100 U/ml

pencillin and 100 lg/ml streptomycin and 1 mM sodium pyruvate.

The cells were maintained at 37�C under an atmosphere of 5%

CO2. Insect Sf9 cells were grown in Grace’s insect medium sup-

plemented with Glutamax, 10% fetal bovine serum, 100 U/ml

pencillin and 100 lg/ml streptomycin at 26�C.

Fluorescence Microscopy

The RINm5F cells were seeded on cover glass chambers

(NUNC, Labdesign, Sweden) on the day before experiments. The

cells were washed with HEPES buffered Krebbs Ringer solution

with 1 g/l glucose (HKRG) and 1 lm Peptide in HKRg was added

and incubated for at least 30 min at 37�C. The cells were washed

twice with HKRg and the cell nuclei were stained with Hoechst

33258 (0.5 lg/ml) for 5 min. The images were obtained with

UltraView ERS confocal live cell imager (PerkinElmer Ltd, Upplands

Vasby, Sweden) connected to a Axiovert 200 (Zeiss, Gottingen,

Germany) and processed in PhotoShop 6.0 software (Adobe Sys-

tems Inc., CA).

Peptide Uptake Detected by Fluorometry

N2a cells were cultured as described above. Stock solutions of

tested peptides were prepared from 1 mM to 5 lM in HKRg

buffer. About 125,000 cells/ml were seeded 2 days before the

experiments in a 12-well-plate, washed and then exposed to 300 llof 5 lM drug in HKRg at 37�C, 30 min, 300 rpm (on thermom-

ixer, Eppendorf). The peptide-treated cells were washed with

trypsin/EDTA (0.05% in HKR), washed thoroughly with HKRg

and lysed in 250 ll 0.1% Triton X-100 in HKR at 4�C for 10 min.

The cell lysates were transferred to a black plate for fluorescence

readout at 492/520 nm. The samples were compared to the fluo-

rescence of the added amount of peptide.

Tissue Preparation

Dissected porcine hearts (260–390 g) were transported from

local slaughterhouse to the laboratory in ice-cold Krebs–Henseleit

solution (119 mMNaCl, 23.8 mMNaHCO3, 3 mMKCl, 1.14 mM

NaH2PO4, 1.63 mM CaCl2, and 16.5 mM glucose). Left anterior

descending coronary artery and great cardiac vein were isolated.

Part of each blood vessel was used immediately after isolation for

contraction tests while part was kept frozen in liquid nitrogen for

the membrane preparation. In some experiments we used blood

vessels in which endothelium was mechanically removed. The same

procedure was used also for the preparation of human umbilical

blood vessels; umbilical cords were obtained from Obstetric Clinic

of Ljubljana Clinical Center, Slovenia.

Blood Vessel Contraction Measurement

The measurements were performed as already described (Ja-

pelj et al. 1999). Rings (5 mm wide) of blood vessels were cut and

mounted into 10 ml tissue chamber filled with Krebs–Henseleit

solution (37�C; pH=7.4) which was oxygenated with the mixture

of 95% O2 and 5% CO2. Initial tension of 50 mN was applied and

after equilibration 30 mM KCl was added to obtain stable iso-

metric contraction. The presence of endothelium was verified with

substance P. After washing out of KCl and substance P and after

the equilibration of the system was restored, test peptide was added

into the tissue chamber and blood vessel contraction was recorded.

1 lM angiotensin and 100 lM M511 were used in order to achieve

the maximal effect of both ligands.

Overexpression of G-proteins in Sf9 Cells

The cDNA for bovine Gas Gai1, Gao and Ga11 in pVL1392

and bovine Gb1c2 in pVL1393 were cotransfected with linearized

baculovirus DNA (Pharmingen, San Diego, CA, USA), and the

resulted virus stocks were subjected to one round of plaque puri-

fication before generation of high-titer virus stocks. In the expres-

sion procedure, Sf9 cells at density of 2 million cells/ml in

suspension culture were infected with a ratio 2:1 of a versus bc.Cells were harvested 48 h post-infection, washed in PBS and stored

at )70� C until membrane preparation.

Membrane Preparation

Frozen pieces of blood vessels were mechanically pulverized.

Subsequently membranes were obtained according to the protocol

of McKenzie (McKenzie 1992) with minor modifications. Until

used they were kept frozen in the concentration of 1–2 mg protein/

ml as determined by the method of Lowry (Lowry et al. 1951). The

same procedure (with the exception of pulverization of tissue) was

used also for the preparation of membranes from Sf9 cells with the

overexpressed G-proteins of different types; protein concentrations

of these membrane preparations were between 0.2 and 0.4 mg/ml.

Measurement of GTPase Activity

The measurements were performed radiometrically according

to Cassel and Selinger (Cassel and Selinger 1976), with the modi-

fications suggested by McKenzie (McKenzie 1992). To the diluted

membranes obtained from Sf9 cells with the overexpressed

G-proteins of different types (final protein concentration in the

assay mixture was 0.01 mg/ml) the ice-cold reaction cocktail containing

ATP (1 mM), 5¢-adenylylimido-diphosphate (1 mM), ouabain

(1 mM), phosphocreatine (10 mM), creatine phospho-kinase

(2.5 U/ml), dithiothreitol (4 mM), MgCl2 (5 mM), NaCl

(100 mM), and trace amounts of [c-32P]GTP to give 50.000–

100.000 cpm in an aliquot of the reaction cocktail (with the addi-

tion of cold GTP to give the required 0.5 lM total concentration of

GTP) was added. Incubation medium was standard TE-buffer, pH

7.5. Background low-affinity hydrolysis of [c-32P]GTP was assessed

by incubating parallel tubes in the presence of 100 lM GTP. Blank

values were determined by the replacement of membrane solution

with assay buffer. GTPase reaction was started by transferal of the

reaction mixtures to 30�C water bath for 20 min. Unreacted GTP

was removed by the 5% suspension of the activated charcoal in

20 mM H3PO4. The radioactivity of the yielding radioactive

Cell-penetrating Protein Mimics 239

phosphate was determined in Packard 3255 liquid scintillation

counter.

The Rate of GTPcS Binding

The binding to G-proteins from blood vessel and cell mem-

branes was followed as described by McKenzie (McKenzie 1992).

Briefly, the membranes (final protein concentration in the assay

mixture was 0.05 mg/ml) were incubated for 3 min in the absence

and presence of peptides in different concentrations with 0.5 nM

[35S]GTPcS at 13�C in TE-buffer (10 mM Tris–HCl+0.1 mM

EDTA), pH 7.5. The unbound [35S]GTPcS was removed by rapid

filtration of the reaction mixture through Millipore GF/C glass-

fiber filters under vacuum. The remaining radioactivity in filters

was determined in the LKB 1214 Rackbeta liquid scintillation

counter. Blank values were determined by replacing the membranes

with buffer.

Islet Isolation

Male Wistar rats, weighing 250–300 g (B&K Universal, Sol-

lentuna, Sweden) were used. The animals had free access to stan-

dard lab chow and drinking water. They were killed by

decapitation, being unconscious after inhalation of carbon dioxide.

The study was approved by the Ethics committee of animal re-

search at the Karolinska Institutet. The islets were isolated by

injecting collagenase A (Sigma Diagnostics Co.) in Hanks’ solution

(9 mg/10 ml) into the pancreas through the pancreatic duct. Then

the gland was removed, incubated for 24 min at 37�C, washed with

Hanks’ solution, and islets were picked up after separation on

Histopaque gradient (Sigma Diagnostics Co.) (Ostenson and Grill,

1986). Isolated islets were cultured over-night free floating in Petri

dishes in RPMI 1640 medium (Flow lab. Ltd.) containing 11 mM

glucose, 2 mM glutamine, 10% heat inactivated fetal calf serum,

100 IU/ml penicillin, and 0.1 mg/ml streptomycin at 37�C, atmo-

sphere 95%O2, 5% CO2.

Batch-Incubation Experiments

After culture, islets were washed and pre-incubated for 30–

45 min at 37�C in 5 ml of Krebs–Ringer bicarbonate (KRB) buffer

containing 118.4 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl2, 1.2 mM

KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3 (equilibrated with

5% CO2 and 95% O2), 10 mM HEPES, 0.2% bovine albumin with

3.3 mM glucose, pH 7.4.

After preincubation, batches of three islets were transferred to

tubes containing 300 ll KRB with glucose concentrations and

substances as indicated. Islets were incubated for 1 h at 37�C in a

shaking water-bath. Three or four tubes were run for each exper-

imental condition. Incubations were stopped by cooling the tubes

on ice. The islets were picked up and aliquots of the media were

stored at )20�C until insulin were measured by radioimmunoassay

with our own anti-porcine insulin antiserum and rat insulin stan-

dard (Herbert et al. 1965).

Inositol 1,4,5-Triphosphate Accumulation

RINm5F cells were seeded 400,000/well in 12-well tissue cul-

ture plates. After 24 h the cells were washed once with PBS and

then incubated with 1 lCi/ml myo[3H]-inositol (Amersham Bio-

sciences, Uppsala, Sweden) in medium 199 (Invitrogen AB,

Stockholm, Sweden) for 20 h, 37�C. Subesquently, the incubation

media was replaced by medium 199, 10 min, 37�C, washed three

times with Hepes Krebs Ringer buffer supplemented with 1 g/l

glucose (HKRg) and incubated with 10 mM LiCl in HKRg, 10 min

37�C. The peptides were added and the cells were incubated 1 h at

37�C. Reactions were stopped by transferring the plates to ice, and

by adding 20% (w/w) ice cold perchloric acid, 30 min on ice. The

supernatant was neutralised with 1.5 M KOH, 75 mM Hepes,

diluted and thoroughly mixed with distilled H2O. Approximately

0.5% of the supernatant was transferred to scintillation vials and

scintillation cocktail was added (Gold Star; CiAB, Stockholm,

Sweden). These vials account for the total amount of accumulated

inositol phosphates. Approximately 90% of the supernatant were

applied to freshly prepared Dowex columns (AG 1-X8; Bio-Rad),

washed with deionised water, and inositol-1,4,5-triphosphate was

eluted with 0.1 M formic acid, 1.2 M ammonium formate into

scintillation vials. All samples were counted in i liquid scintillation

counter.

Curve fittings and other calculations as well as graphical pre-

sentations of the results were done by Prism computer package

(GraphPad Software Inc., USA).

RESULTS

Design of Peptides

The amino acid sequences for M511, G53-1 andG53-2 have been selected using the CPP predictionprogram (Hallbrink et al. 2003, 2005). In short, themethod of identifying a CPP, according to this ap-proach, comprises of assessing the averaged bulkproperty values of the amino acids of the definedsequence, and ensure that they fall within the bulkproperty value interval obtained from a training setof known CPPs. M551 is the scrambled version ofM511 (Table I). G53-2 is a modified version of G53-1, covering a more N-terminal part of the thirdintracellular loop of the GLP-1R, and with sequencealterations rendering the peptide more CPP-likeproperties according to the prediction program(Table I).

Synthetic peptides derived from natural proteinshave been a useful tool in studies of protein functionand protein–peptide interactions. However, this ap-proach has been limited by delivery of peptide to thetarget-location, allowing studies only on membranepreparations. We have found sequences within pro-teins bearing CPP-properties (Fig. 1), and peptidesbased on these sequences may be useful in functionalstudies of the parent protein in living cells and tissues.Moreover, the peptides open up novel ways forreceptor targeted drug design.

240 Ostlund et al.

Translocation of Peptides into Cells

All three peptides, M511, G53-1 and G53-2translocate into live cells and at levels comparable tothe well characterized CPP pVEC (Fig. 1). The livecell uptake visualized by microscopy is performed onRINm5F cells (Fig. 1a–d). In the quantitative uptakeassay, N2a cells were used (Fig. 1e). G53-1 is a CPP,despite the low uptake (Fig. 1e), since it is clearlytranslocated into cells (Fig. 1c).

M511 Causes Contraction of Blood Vessels

M511, but not the scrambled version, M551, pro-motes intensive and long-lasting contraction of por-cine left anterior descending coronary artery (Fig. 2a)in a concentration-dependent manner (Fig. 2b). Thestrength of contraction is comparable to the effect of30 mM KCl, which is approximately 50% of maximaleffect ofKCl.With higher concentrations, themaximaleffect approaches the effect of 90 mM KCl, which isgenerally considered to be the maximal attainablecontraction effect (Japelj et al. 1999). Contrary to thecontractory effect of KCl, the effect ofM511 could notbe terminated by washing M511 out of the assaysolution and it also showed a concentration-dependent5–15 min lag-period after addition of M511 beforecontraction occurred. Our results (Fig. 2a, middlepanel) also show that blood vessel endothelium is notrequired for the effect of M511. Similar blood vesselcontracting effects ofM511were found inporcine greatcardiac vein and human umbilical vein (data notshown).

Involvement of Signaling Pathways in M511-Induced

Vessel Contraction

U73122, a phospholipase C inhibitor, effectivelyreduces the blood vessel contraction if applied priorM511 (Fig. 2c). We have also used the angiotensin IIantagonist Sar1-Thr8-angiotensin II in order to seewhether or not the mechanisms of blood vessel

Table I. Presentation of the Peptides; Names, Original Proteins and Sequences (r=rat, m=murine)

Name Original protein Sequence

M511 R/m AT1AR (304-318) FLGKKFKKYFLQLLK-amideM551 Scrambled sequence of M511 KGKFQLYLKLKFKFL-amideG53-1 rGLP-1R (330-351D344–346) IVIAKLKANLMCKTCRLAK-amideG53-2 rGLP-1R (316-337, F321Y, I323V, K323-324ins, R326K, C329A) AIGVNYLVKFIKVIAIVIAKLKA-amidepVEC mVE-cadherin (615–632) LLIILRRRIRKQAHAHSK-amide

Fig. 1. Cellular uptake of carboxyfluorescein labeled peptidesM511, M551, G53-1 and G53-2. Fluorescence microscopy of (a)M511 (b) M551 (c) G53-1 (d) G53-2 in RINm5F cells. (e) Quanti-tative detection of intracellular fluorosceinyl labelled peptides. Theexperiments were performed in N2a mouse neuroblastoma cells,incubated for 30 min at 37�C at 5 lM concentration of respectivepeptide. pVEC was used in the same assay for comparison.

Cell-penetrating Protein Mimics 241

contraction induced by M511 and by angiotensin IIare different (Fig. 2d). The contraction effect ofangiotensin II on the porcine left anterior descendingcoronary artery was reverted by the antagonist, butthere was no effect on the M511-induced contractionof blood vessels. Our results also show that M511 ismore efficient than angiotensin II, since it inducedabout 40% higher contraction force compared to thatinduced by 30 mM KCl, while the effect of angio-tensin II was almost the same.

In order to shed some light on the type of G-proteins that were affected by M511, we used mem-branes from Sf9 cells overexpressing G-proteins ofdifferent types, and measured the GTPase activity ofthese membranes in the absence and presence ofM511 (100 lM). The results summarized in Fig. 3ashow a small but significant activation of Gi and Go

and, interestingly, an inhibition of G11. Gs type of G-proteins was not affected. Fig. 3b indicates thatGTPcS binding is accelerated by the action of M511

in a dose-dependent manner; M551 did not show anyeffect. Experiments were performed on membranesfrom porcine left anterior descending coronary artery.The EC50 of M511 was found to be of �0,1 mM.

Effect of G53 Peptides on Insulin Release

Treatment of isolated rat pancreatic islets with10 lM G53-2 showed a statistically significant in-crease of insulin release both at low and high glucoselevels (Table II). The effect under high glucose con-ditions was not additive with the effect of receptor-saturating concentrations of GLP-1, indicating thatthis peptide shares a common signaling pathway withthe receptor. In contrast, at high glucose concentra-tions, G53-1 showed an inhibition of insulin release,which was affected by addition of GLP-1 (Table II).G53-1 at low glucose concentration had no effect oninsulin release (Table II). The effects of the G53peptides were dose-dependent (data not shown).

Fig. 2. M511 induced blood vessel contraction and involvement of PLC pathway. (a) Effect of M511 (upper and middle panel) and M551(lower panel) on the contraction of the porcine left anterior descending coronary artery without epithelium (upper panel) and in the presenceof intact epithelium (middle and lower panel). Contraction force is given in the arbitrary units. 1 – administration of 30 mM KCl, 2 –administration of substance P, 3 – washing-out, 4 – administration of 16 lMM511 or M551. (b) Effect of M511 on maximal observed relativecontraction of porcine coronary artery (n). Contraction obtained by 90 mM KCl was taken as 100%. Points are averages of four independentexperiments. (c) Effect of PLC inhibitor on the contraction of the porcine left anterior descending coronary artery induced by M511. 1 –30 mM KCl, 2 – substance P, 3 – buffer wash, 4 – 16 lM M511, 5 – 30 lM U73122. (d) Effect of Sar1-Thr8-angiotensin II on the contractionof the porcine left anterior descending coronary artery induced by M511 (upper graph) and angiotensin II (lower graph). 1 – 30 mM KCl, 2 –buffer wash, 3 – 100 lM M511, 4 – 1 lM angiotensin II, 5 – 45 lM Sar1-Thr8-angiotensin II.

242 Ostlund et al.

Hence, G53-1 inhibited insulin release at high glucoselevels and does not seem to share the same signalingpathway as the receptor. G53-2, on the other hand,stimulated insulin release and does seem to share thesignaling pathway with the receptor.

Involvement of Signaling Pathways in G53-induced

Insulin Release

G53 peptides stimulate GTPcS rate of binding toSf9 membranes enriched with Gs, Go, Gi1 and G11

type of G-proteins, albeit G53-2 to a much higherextent than G53-1 (Fig. 4a). Binding to G11-enrichedmembranes was not affected by G53-1, whereas G53-2 heavily influenced the rate of binding (5 times overbasal levels). Therefore, for confirmation, we per-formed IP3 measurements on rat insulinomaRINm5F cells. Accordingly, G53-1 caused none orvery little IP3 production, both at low and high glu-cose concentrations. G53-2, on the other hand, elic-ited 500% higher levels of IP3 as compared to control,at low and high glucose concentrations. These resultsmatch nicely with the results on insulin release, aswell as with the results from GTPcS binding.

DISCUSSION

M511, the peptide corresponding to rat andmouse AT1AR positions 304-318 (Table 1), was ob-served to act as a CPP and as a powerful contractorof blood vessels. As such, it is an interesting drugcandidate aimed against chronic hypotension andpossibly also migraine. Its potential disadvantage isthe relatively high concentration (16 lM) requiredfor vessel contraction but on the other hand itinternalizes spontaneously into the cells and has along-lasting effect. Since M511 is derived from theintracellular C-terminal part of AT1AR it seemspossible that its mechanism of action is based onuncoupling of the receptor from G-proteins. Theobserved lag-period (Fig. 2a) could be interpreted asthe time required for the penetration of sufficientamount of the peptide into the cell, thus, the inabilityto terminate the contraction by washing M511 from

Fig. 3. Effect of M511 on G-proteins. (a) Relative GTPase activityof membranes prepared from Sf9 cells overexpressing Gi, Go, Gs,and G11 heterotrimeric G-proteins, respectively. Basal GTPaseactivity in each type of membranes in the absence of M511 was setto 100%. (b) Rate of GTPcS binding to membranes prepared fromthe porcine left anterior descending coronary artery. (d – nopreincubation of membranes with M511; s – 25 min preincubationof the membranes with M511, and � – no preincubation ofmembranes with M551). Points are averages of four independentexperiments.

Table II. Effects of G53-1, G53-2 and GLP-1 on Insulin Release from Isolated Rat Pancreatic Islets in Low and High Glucose Conditions(Results are Presented as lU insulin/islet/h)

Peptide Glucose (lM) Control (lU insulin/islet/h) 10 lM peptide (lU insulin/islet/h)

G53-1 3.3 11.3±1.2 12.6±0.8G53-2 3.3 8.7±1.4 31.5±6.8**G53-1 16.7 65.0±14.5 41.3±5.5*+GLP-1 112.5±8.4 84.3±8.8G53-2 16.7 62.8±6.6 103.8±10.4**+GLP-1 105.1±15.5 109.9±5.3

GLP-1 was added at 100 nM. Results are given as mean±SEM of 5–6 experiments (*P<0.05, ** P<0.01, *** P<0.001 vs without peptide atsame glucose concentration).

Cell-penetrating Protein Mimics 243

the assay solution, would be in accordance with itsintracellular localization. Virtually identical effects ofM511 in porcine artery and great cardiac vein andhuman umbilical artery, illustrate that this effect isnot restricted only to arteries and indicate a generalfunction in blood vessels of different tissues.

In order to study the signaling pathways involvedin M511-induced blood vessel contraction, we usedthe phospholipase C inhibitor U73122. This com-pound is able to penetrate cells due to its steroid-likestructure and to effectively inhibit phospholipase C inthe lM range (Bleasdale et al. 1990; Yule andWilliams 1992). U73122 did not affect tonus of theporcine left anterior descending coronary artery(Fig. 2c, middle panel), nor did the inhibitor modifyblood vessel contraction induced by M511, whenadded after the peptide (Fig. 2c, upper panel), but itsubstantially decreased (more than 50%) the effect of

M511 when added 30 min prior to M511 (Fig. 2c,lower panel). This indicates that blood vessel con-traction by M511 is mediated via the inositol phos-phate mechanism.

Furthermore, we have found that M511 triggerscontraction independently of angiotensin II and itsreceptors, since the angiotensin II antagonist Sar1-Thr8-angiotensin II was unable to affect the con-traction of blood vessels induced by M511 (Fig. 2d).This corroborates our finding that M511 does notactivate AT1AR but instead probably binds directlyto G-proteins and induces activation of phospholi-pase C. Moreover, M511 is more efficient thanangiotensin II in contracting the blood vessels, sinceit induced about 40% higher contraction force com-pared to that induced by 30 mM KCl, while the effectof angiotensin II was almost the same as the effect of30 mM KCl.

In order to investigate which G-proteins are in-volved in the action of M511, GTPase activity in Sf9membranes overexpressing different types of G-proteinswas measured (Fig. 3a). Slight activation of Gi/Go andno effect on Gs is in good accordance with previousstudies. An explanation for the inhibition of G11

instead of its activation could be that G11 contrary toGi and Go (see also Shirai et al. (1995)) behavessensitively to the presence or absence of the thirdintracellular loop of AT1AR. Unfortunately, Sf9 cellsoverexpressing Gq-proteins have not been available inour laboratory, therefore we did not test the effect ofM511 on this type of G-proteins. However, Sanoet al. have found that peptides corresponding toamino acids 306-320 of AT1AR with mutations in theC-terminal part seriously affects the coupling to, andactivation of, Gq (Sano et al. 1997). In line with this,Tang et al. have found that the region 310–318 ofAT1AR is required for Gq activation (Tang et al.1998). All this taken together, we can assume thatM511 also might interact with Gq type of G-proteins.

As presented in Fig. 3b, the rate of binding ofGTPcS to the membranes obtained from the porcineleft anterior descending coronary artery was dose-dependently increased by M511. The upper plateau ofthe effect was not obtained since concentrations over500 lM could not be used. It is obvious that thiseffect is specific for M511 since the scrambled peptideis not active, although it is taken up by live cells.Similar results were obtained with membranes pre-pared from porcine great coronary vein and humanumbilical artery (data not shown). The increased rateof GTPcS binding indicate the involvement ofG-proteins in the process of blood vessel contraction

Fig. 4. Effects of G53 peptides on GTPcS binding and IP3 pro-duction. (a) GTPcS binding of G53-1 and G53-2 (100 lM) tomembranes prepared from Sf9 cells overexpressing Gi, Go, Gs, andG11 heterotrimeric G-proteins, respectively. Bars represent averagevalues of three independent experiments. (b) IP3 production inRINm5F. Low glucose=3.3 mM, high glucose=16.7 mM, 10 lMpeptide was added.

244 Ostlund et al.

in the presence of M511 and corroborate the idea thatM511 uncouples G-proteins from the AT1AR. It iswell known that any uncoupling of G-proteins fromthe 7-transmembrane receptors generally results inincreased rate of GTPcS binding and in increasedGTPase activity (McKenzie 1992). The presentedresults match well with the effect of the peptides onblood vessel contraction (Fig. 2), and are in accor-dance with the suggestion that AT1ARs function viainhibition of adenylyl cyclase (activation of Gi/Go)and via modulation of phosphoinositide metabolism,most probably through pertussis toxin insensitiveG-proteins, most probably Gq and G11 (Touyz andBerry 2002). We have indeed demonstrated regula-tion of G11 by M511, however, not by activation ofthis type of G-proteins but by their inhibition. Al-though we have not demonstrated its selectivity toAT1 mediated signal transduction, we show that thepeptide most probably activates the same type ofG-proteins as agonist activated AT1AR.

We have also performed a shorter study on twopeptides derived from the third intracellular loop ofthe GLP-1R, G53-1 and G53-2. G53-2 internalizedinto cells to a higher extent than G53-1 (4 timeshigher) (Fig. 1), an expected result, since the aminoacid sequence of G53-2 had been altered with the aimto achieve a peptide with pronounced cell-penetrablefeatures. As the GLP-1R is involved in glucosestimulated insulin release, we performed studies ofinsulin release on isolated rat pancreatic islets incu-bated with the peptides. At low glucose levels, G53-2induced release of insulin at levels approximately 4times higher than control levels. G53-1 had no effectunder these conditions and this could be explained bythe differences in cellular uptake between the twopeptides. To mimic a postprandial situation, thepeptides were also added at a high glucose concen-tration. Also under these conditions, G53-2 stimu-lated insulin release, whereas G53-1 was shown to beinhibitory. By adding receptor saturating concentra-tions of GLP-1, we could deduce that G53-2 andGLP-1 probably share the same signaling pathway,while G53-1 and GLP-1, due to their additive effect,do not.

Glucose-induced insulin release mediated via theGLP-1R has been found to be mediated throughseveral different signaling pathways, including sig-naling through phospholipase C, adenylyl cyclase andphosphatidyl inositol 3-kinase (Goke and Conlon1988; Goke et al. 1989; Dillon et al. 1993; Lu et al.1993; Wheeler et al. 1993; Widmann et al. 1994;Galera et al. 1996; Marquez et al. 1998; MacDonald

et al. 2002). After studying the effect of the G53peptides on GTPcS binding, we saw effects of bindingwith G53-2 on all G-protein subtypes tested (Gi1, Go,Gs and G11), which is in accordance with previousstudies on GLP-1. G53-1 had a slight effect onmembranes overexpressing Go and Gs. This was aninteresting finding, considering our conclusion thatthe differences in insulin release between G53-1 andG53-2 was due solely to the differences in the abilityto translocate into cells. In the GTPcS bindingexperiment, which is performed on cellular mem-branes, the cell penetrable features of a peptide is ofno interest. Consequently, this implies that G53-1 notonly is a weak CPP, but also that it would probablyhave little effect even if it was translocated into thecell.

The biggest difference in effect between G53-1 andG53-2 was on Sf9 membranes overexpressing G11.Subsequent IP3 measurements revealed that actionsof G53-2 is, at least in part, mediated through theinositol phosphate pathway. The signaling leading tothe inhibitory effect of G53-1 remains to be revealed,but at this point we can rule out the involvment ofG11/inositol phosphate.

Previous studies supporting our results are e.g.experiments performed by Bavec and coworkers,showing that the third intracellular part of the GLP-1R stimulates Gi1, Gs, Go and G11 as demonstratedby using a peptide corresponding to residues 329–351in GTPcS binding studies (Bavec et al. 2003).Moreover, point mutations in this region have re-vealed that the third intracellular loop is responsiblefor the interaction with G-proteins (Heller et al. 1996;Takhar et al. 1996; Mathi et al. 1997; Bavec et al.2003).

In conclusion, we have demonstrated that twonovel CPPs, derived from the intracellular parts ofGPCRs, can act as cell-penetrating peptides, as wellas mimic the interaction of the GPCR and G-proteinin the target cell. This opens novel possibilities in thedesign of intracellular effectors through the detectionof cell-penetrating, bioactive sequences in differentsignaling proteins.

CONCLUDING REMARKS

The present study reports three peptides, whichoriginate from G-protein coupled receptors. Ex vivothese peptides trigger identical physiological effect aswould do agonist activations of respective receptors.The peptides are shown to penetrate cell membranes,

Cell-penetrating Protein Mimics 245

interact with the same types of G-proteins as originalreceptors and trigger respective intracellular signalingcascades. M511, a 16-amino acid fragment of angio-tensin AT1A receptor was shown to cause bloodvessel contraction. G53-1 and G53-2 originating fromglucagon-like peptide receptor affected insulin releasefrom isolated pancreatic islets.

Our results demonstrate that short cell-pene-trating peptides can not only be used as deliveryvehicles. Cell-penetrating sequences derived fromnatural proteins, can have specific intracellular ef-fects, mediated by peptide–protein interactions.Additionally, commonly synthetic peptides act asdecoys inhibiting interactions of parent proteins.Peptides presented here act as activators of certainG-proteins subtypes. This opens a new possibilityfor studying receptor functions, protein-proteininteractions and intracellular signal transduction. Infurther terms such peptides may prove useful inmedical applications, where upregulation of a spe-cific physiological response, like vasoconstriction orinsulin release is desired.

ACKNOWLEDGEMENTS

We are grateful to Mr. Joel Ohlsson for the assistance with

peptide synthesis, Dr. Ziva Novak-Antolic for providing human

umbilical cords and Dr. Carl-Johan Dalsgaard for fruitful discus-

sions. This work was supported by grants from European Com-

munity (QLK3-CT-2002-01989), Swedish Research Council VR-

NT, CePeP AB, Stockholm, Sweden, Orexo AB, Uppsala, Sweden

and the Ministry of Science and Education, Slovenia. The pre-

sented results are protected by the international patent application

with international publication number WO 03/106491 A2.

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