Cellular Uptake and in Situ Binding of a Peptide Agonist for Calmodulin

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Biochemical and Biophysical Research Communications 277, 462–469 (2000)

doi:10.1006/bbrc.2000.3691, available online at http://www.idealibrary.com on

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ellular Uptake and in Situ Binding of a Peptidegonist for Calmodulin

ichael K. Manion,* Matteo Villain,† Z. George Pan,‡ Jay M. McDonald,‡ and J. Edwin Blalock§,1

Fred Hutchinson Cancer Research Center, Seattle, Washington; †Department of Biochemical Medicine, Universityf Geneva, Geneva, Switzerland; and ‡Department of Pathology and §Department of Physiology and Biophysics,niversity of Alabama at Birmingham, Birmingham, Alabama

eceived September 13, 2000

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We have used the method of inverted hydropathy toevelop peptides that interact with EF hands of cal-odulin (CaM). Previously we have shown these pep-

ides specifically interact with their desired target in aroductive manner, in that they activated CaM in thebsence of Ca21. Therefore, we sought to determinehether these peptides would enter cells, remain in-

act, and interact with CaM in the interior of the cell.sing several techniques we have demonstrated cellu-

ar uptake, stability, and an intracellular interactionith CaM with fluorescein-labeled and radiolabeledeptides in Jurkat T cells. The results suggest thathese peptides may be useful in the study and theanipulation of Ca21-mediated pathways in cells.

2000 Academic Press

Key Words: complementary peptide; EF hands; hy-ropathy; Ca21 pathways.

Ca21 is a ubiquitous second messenger within cellsnd plays an important role in many physiologic andathophysiologic signaling cascades resulting in mul-iple biological outcomes. The interaction of Ca21 withhese pathways is often mediated by the protein cal-odulin (CaM) which has four Ca21 binding sites,

ermed EF hands. Upon the coordination of Ca21 by theoop sequences of the EF hands, a conformationalhange occurs in CaM thus exposing binding sites forany CaM binding peptides and proteins. The ability

o design and synthesize molecules that interact withaM and modulate its activity is of great benefit notnly for the study of Ca21-mediated signals but also forhe potential development of compounds to impedeathophysiological conditions arising from inappropri-te Ca21 signaling.

1 To whom correspondence should be addressed at University oflabama at Birmingham, Department of Physiology and Biophysics,918 University Blvd. MCLM 896, Birmingham, AL 35294-0005.ax: 205-934-1446. E-mail: [email protected].

462006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ave designed a series of peptides targeted to interactith the loop sequence of the EF hands, and we term

hese peptides Calcium Like Peptides (CALP) (3, 4).e have demonstrated in vitro a specific interaction ofALP with EF hands of troponin C as well as CaM.he peptide/CaM complex displayed a conformationalhange related to that induced by the co-ordination ofa21 by CaM, and the CALP/CaM complex was able toctivate phosphodiesterase (PDE) in the absence ofa21. These results demonstrated a specific and pro-uctive interaction of CALP with the desired targetrotein (3, 4). Due to their overall hydrophobicity andize, we hypothesized that CALP could enter cells,ince several other peptides of similar size and hydro-hobicity have also been demonstrated to gain accesso the interior to cells (5–10). Indeed, we found thathese peptides are able to enter the cell, and directly,s well as indirectly, through CaM, block the activity oflutamate receptor channels in cultured neocorticaleurons, as well as a non-selective cation channel,resent in Jurkat T cells, which is activated by HIV-1p120. As a consequence, apoptosis mediated by annflux of Ca21 through these channels was also doseependently inhibited by these novel peptides (11).In the present report, we have sought to gain a better

nderstanding of the peptide/cell interactions of thisovel type of Ca21 channel regulator. In particular, weave studied the cellular uptake process, stability, andubcellular distribution of CALP and provide biochem-cal evidence for its in situ interaction with CaM.

ATERIALS AND METHODS

Peptides. All peptides used (see Table 1 for sequences) were syn-hesized in our laboratory using flow solid phase peptide synthesisith (N-(9-fluorenyl)methoxy-carbonyl) chemistry and O-pentafluo-

ophenyl ester (Opfp) amino acids activated with HOAt (1-hydroxy--azabenzotriazole), on a PerSeptive 9050 Peptide synthesizer. Alleagents for peptide synthesis were purchased from PerSeptive Bio-ystems (Framingham, MA) unless otherwise mentioned. Pre-loadedEG-PS (polyethylene glycol graft polystyrene) resin was used, and

all the peptides were purified by reverse phase-high performancel3o0Ts

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Vol. 277, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

iquid chromatography (RP-HPLC) on a Delta Pack C18 300A 30 3id cm column. The purity of the product was checked by RP-HPLC

n a Dynamax C18 (300 3 4.8 id mm) column with a gradient fromto 90% Acetonitrile and 0.1% trifluoroacetic acid (TFA) in 40 min.he identity of the peptides was confirmed by TOF-MALDI masspectrometry (University of Alabama at Birmingham Core Facility).For F-CALP1 (Table 1) the sequence was extended in the carboxyl-

erminal direction with a cysteine. The side chain sulfur was selec-ively functionalized using fluorescein-5-malemide (Pierce, Rockford,L) at pH 6.5. The final product was purified by RP-HPLC andonfirmed by mass spectroscopy.Selectively radiolabeled CALP1 (14C-CALP1; Table 1) was synthe-

ized inserting N-Fmoc-Valine, [1-14C]-OH (American Radiolabeledhemicals, St. Louis, MO) in position 1 of the peptide. The labeledmino acid was coupled to resin containing the first seven aminocids of the sequence in a 1:10 ratio of amino acid/resin amino acidroups. The activating agent was HATU/DIPEA. After 2 h, an excessf unlabeled Fmoc-Val-OH/HATU/DIPEA was added to the reactionessel. The Fmoc was removed with 20% piperidine, and the peptideas cleaved with 95% TFA and 5% H2O. The cleaved peptide wasrecipitated in cold ether and lyophilized. The specific activity was.42 3 106 cpm/mg of solid peptide. Peptides were stored as lyophi-ized 50 mL aliquots of a 5 mM stock at 220°C, reconstituted in

illiQ water before being diluted in buffer or media at final concen-rations shown.

Cells and cell culture. Jurkat E6-1 T cells (American Type Cul-ure Collection, Norcross, GA) were cultured at 37°C with 5% CO2 inPMI media with 10% Fetal Bovine Serum (Atlanta Biologicals), 100g/mL penicillin/streptomycin, (13) nonessential amino acid solu-

ion, 2 mM L-glutamine, 1 mM sodium pyruvate (Cell-Gro, Herndon,A), 23.8 mM sodium bicarbonate, 10 mM N-(2-Hydroxyethyl)-iperazine-N9-(2-ethanesulfonic acid) (Hepes) (Sigma, St. Louis,O). Jurkat cells were diluted 1:10 in fresh media every 2 to 3 days.

table CaM-expressing Jurkat cells, prepared as described below,ere grown in the same media, with the addition of 100 mg/mlygromycin (Sigma, St. Louis, MO) and 100 mg/ml geneticin (Sigma).orty-eight hours prior to experiments, these cells were also incu-ated with various concentrations of tetracycline (Tet), which dose-ependently reduced the expression of CaM.

Preparation of stable CaM-expressing Jurkat cells. A tetra-ycline-off (Tet-off) gene expression system was used to transfecttable human CaM sense constructs into Jurkat T lymphocytes. Theet-off expression plasmid (pTRE) was modified by inserting a 1.4 kbygromycin resistant gene fragment from the pTyg vector (Clontech,alo Alto, CA). A full length 0.9 kb human CaM gene was digested

rom pBluescript-CaM cDNA (ATCC) with Xho1 endonuclease.eanwhile the modified pTRE plasmid was digested with Sal I

ndonuclease. The 0.9 kb Xho-I–Xho-I restriction fragment contain-ng the CaM gene was subcloned into a Sal-I digested modified pTRElasmid. The orientation of the CaM inserts was ascertained by DNAequence analysis. Normal Jurkat T cells were transfected with theTRE expression vector containing the sense CaM cDNA by electro-oration in a Bio-Rad Gene Pulsar at 960 mF and 0.22 kV/cm (t 55–30 ms). Selection of stable Tet-off expression cell lines was initi-ted 48 h after transfection with 100 mg/ml geneticin, 300 mg/mlygromycin, and 2 mg/ml tetracycline in RPMI1640, and completeedium was replaced every 4 days. After 5 to 7 days, living cells were

eparated from dead cells and plated at a lower density. After serialilution, isolated single cell clones were cultured in 96-well platesnd then transferred into 12-well and 6-well plates and then into a5 cm T flask.

Fluorescent microscopy of F-CALP1 loaded Jurkat T lymphocytes.pproximately 50 mM F-CALP1 was added to the culture media, and

he cells were incubated at room temperature in the dark for 15 min.hen Hoechst 33258 (7.5 mM, Sigma) was added, and the cells were

ncubated for a further 5 min. This was followed by washing the cells

463

ith normal Ringer’s solution, and fixing for 5 min at room temper-ture with Bouin’s solution (Sigma). The cells were then washedwice with Ringer’s solution and mounted on glass slides. Digitalmages were acquired using an Olympus IX70 digital confocal mi-roscope (Lake Success, NY) equipped with a Sensys 1400 CCDamera (Photonics, Tucson, AZ). Image analysis was done usingPLab Spectrum software.

Uptake of 14C-CALP1 into Jurkat T lymphocytes. Jurkat T cellsere firstly passed over a ficoll gradient to remove dead cells, washed

wo times in fresh RPMI (serum free), and resuspended in 400 ml ofPMI at a concentration of 2.5 3 107 cells/ml. 14C-CALP1 was addedt the specified concentration, from a stock solution of 5 mM, and theells were then incubated for the specified time at 37°C. After incu-ation, the cells were placed on ice and transferred to a 4°C coldoom, where the rest of the procedure was conducted. The cells wereentrifuged for 1 min at 1,000g and then resuspended in 100 ml ofold RPMI supplemented with 1% bovine serum albumin (BSA) thatad been adjusted to pH 2.8 with HCl. Cells were incubated in thisedium for 3 min, which was then neutralized by the addition of 900l of cold RPMI supplemented with 100 mM Hepes, pH 7.2. The cellsere then centrifuged for 1 min at 1,000g and washed two more

imes in this medium. The cells were either used for other procedurest this point or directly counted on a Tri-Carb 2200CA liquid scin-illation analyzer (Packard, Meridan, CT). Samples from each washere also counted at the end of each experiment.

Subcellular fractionation of Jurkat cells. Subcellular fraction-tion of Jurkat cells into membrane, cytosolic, and nuclear fractionsas accomplished by firstly lysing cells in a buffer containing (inM) 10 Hepes, 10 NaCl, 1 KH2PO4, 5 NaHCO3, 1 CaCl2, 0.5 MgCl2,ethylendediamine tetraacetic acid (EDTA), and 1% mammalian

rotease inhibitor cocktail (Sigma). This was then dounce homoge-ized 50 times and centrifuged at 7,500 rpm for 5 min at 4°C. Thisrude nuclei pellet was then purified in 1 ml buffer containing (inM) 10 Tris, 300 sucrose, 1 EDTA with 0.1% NP-40, and 1% mam-alian protease inhibitor cocktail. This was dounce homogenized a

urther 30 times and centrifuged at 5,000 rpm for 5 min. The mem-rane and cytosolic fractions were prepared by centrifuging theupernatant from the first spin, at 100,000g for 30 min.

HPLC analysis. Cytosolic fractions of Jurkat cells loaded with4C-CALP1 were analyzed by RP-HPLC on an ABI aquapore C830 3 2.1 id mm) column with a gradient from 0 to 90% acetonitrilend 0.1% TFA in 40 min. Fractions were collected from each runvery minute and counted by liquid scintillation for the presence ofadioactivity associated with the peptide. This was compared toetention times for unlabeled peptide, 14C-CALP1 alone, and 14C-ALP1 which had been mixed with the cytosolic fraction of Jurkatells. No change in retention time was observed in any of theontrol runs.

Crosslinking with DSS. Jurkat cells or purified bovine brainaM were first incubated with 14C-CALP1 for 10 min, and cells were

Amino Acid Sequences of CALP and Control Peptides

Name Sequence

CALP1 VAITVLVKF-CALP1 VAITVLVKC(Fluorescein)F-MBP (Fluorescein)CASQKRPSQR14C-CALP1 ([1-14C]V)AITVLVK

Note. Peptides shown from amino- to carboxy-terminal. The fluo-escein maleimide on F-CALP1 and F-MBP is linked through theulfhydryl group on the cysteine. ([1-14C]V) refers to valise, [1-14C]nserted in position 1 of 14C-CALP1.

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cid stripped and washed as described above, except that the finalash and resuspension was in cold phosphate buffered saline (PBS).o this, 1 mM DSS (Pierce, Rockford, IL) was added, and the cellsere incubated at 4°C for 1 h. The crosslinking was then quenchedy the addition of 100 mM Tris for 15 min. After this, the cells wereashed three times in cold PBS.

Immunoprecipitation. Cells crosslinked in this way, as well asontrols, were lysed in 1 ml of a PBS buffer containing 1% Nonidet,.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1%ammalian protease inhibitor cocktail. Each cell sample was then

isrupted by aspiration through a 21 gauge needle several times, andhey were passed to a separate tube. The original sample tubes werehen washed with an additional 1 ml of this buffer. Cellular debrisas then pelleted by centrifugation at 3,000 rpm at 4°C for 15 min.rotein was assayed (BCA, Pierce) in the supernatants, and each cellample was standardized to 150 mg total protein/ml. The remainingamples were then split into two 1 ml groups, one of which wasncubated with 2 mg goat-anti-CaM and the other was incubated withormal goat immunoglobulin G (IgG) (Santa Cruz Biotechnology,anta Cruz, CA) overnight at 4°C with gentle mixing. To this wasdded 30 ml Protein-A/G conjugated sephadex beads (Santa Cruz

FIG. 1. Fluorescent microscopic images of Jurkat T lymphocytes lytoplasmic staining (green) throughout the cell. Cells were counters) No cytoplasmic staining was noticed with the fluorescein-labeledm for A and B and 25 mm for C and D.

464

iotechnology), and the tubes were incubated overnight at 4°C withentle mixing. Immunoprecipitates were collected by centrifugationt 2500 rpm for 5 min at 4°C. Supernatant was collected, and theellet was washed 4 times with PBS. Samples from both the un-ound (supernatant) as well as the bound (pellet) were then countedn the liquid scintillation analyzer. Data are represented as % bound/otal (i.e., bound 1 unbound) 6 SD, n 5 3 groups. Samples of cellssed in this experiment were also analyzed by Western blotting toetermine relative levels of CaM expressed.

Western blotting. Samples of lysed Jurkat cells (10 mg total pro-ein as determined by BCA assay, Pierce) were subjected to SDS–oly acrylamide gel electrophoresis (PAGE) (15% acrylamide gels),nd then transferred to polyvinylidene difluoride (PVDF) mem-ranes under normal conditions. After transfer, blots were fixedvernight in tris buffered saline (TBS) with 0.2% glutaraldehyde tox CaM to the membrane. Blocking was done for 2 h at roomemperature with 5% milk powder in TBS-tween 20 (0.1%, TBS-T).ncubations with the primary reagent, highly specific monoclonalouse anti:CaM antibody (12), were conducted at room temperature

or 2 h in TBS-T supplemented with 1% BSA. Incubations with theecondary antibody (AP-conjugated rabbit anti:mouse) were done at

ed with F-CALP1. (A, C) Jurkat T cell stained with F-CALP1 showedned with Hoechst 33258 (7.5 mM, blue color) to show the nuclei. (B,rophilic control peptide, F-MBP. White bars on figures represent 4

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oom temperature for 1 h in TBS-T with 5% milk powder. The blotas then developed using Bio-Rad AP substrate kit, using manufac-

urer’s instructions. After development and flatbed scanning, blotsone with 14C-CALP1 were placed in a cassette with Kodak BiomaxS double emulsion film and exposed at 280°C for 6 weeks before

rocessing.

ESULTS AND DISCUSSION

Our first approach to demonstrate the ability ofALP to enter cells involved the synthesis of a modi-ed version of CALP1 (see F-CALP1 in Table 1) whichas conjugated to fluorescein malemide so that it coulde visualized by fluorescent microscopy. Fluorescenticroscopy revealed that F-CALP enters Jurkat T cells

Figs. 1A and 1C show representative cells incubatedith F-CALP1 and Hoechst 33258) resulting in stain-

ng throughout the cytoplasm and nucleus. A hydro-hilic control peptide of the same size was also conju-ated to fluorescein malemide (see F-MBP in Table 1),hich did not enter cells (Figs. 1B and 1D). This dem-nstrates that the fluorescein malemide is not respon-ible for uptake of CALP by the cells. However, sincehe conjugation of the peptide with the fluorescein

FIG. 2. Uptake of 14C-CALP1 by Jurkat T lymphocytes. 107 cellser condition were incubated with 100 mM 14C-CALP1 (i.e., 2.61 305 cpm/sample) at 37°C for 10 min and then immediately trans-erred to 4°C, where they were acid stripped and then washed threeimes. Before the final spin cells loaded with peptide were thenivided into three groups, one of which was counted immediately,ne was kept at 4°C for 30 min before the final spin and counted, andhe third was kept at 37°C for 30 min before the final spin andounted. White bars represent the total radioactivity in the finalample; gray bars represent radioactivity in the fraction containinghe cells, while black bars represent radioactivity present in the finalash. Each condition was done in triplicate; mean 6 SD shown.

465

alemide might alter the activity of the peptide, wesed a radiolabeled version of CALP1 (14C-CALP1) forhe following experiments.

FIG. 3. Dose response of the uptake of 14C-CALP1 by Jurkat Tymphocytes. 107 cells per condition were incubated with variousoncentrations of 14C-CALP1 at 37°C for 10 min and then immedi-tely transferred to 4°C, where they were acid stripped and thenashed three times before being counted. Each condition was done in

riplicate; mean 6 SD shown.

FIG. 4. Kinetics of the uptake of 14C-CALP1 by Jurkat T lym-hocytes. 107 cells per condition were incubated with 100 mM 14C-ALP1 at 37°C for different periods of time and then immediately

ransferred to 4°C, where they were acid stripped and then washedhree times before being counted. Each condition was done in tripli-ate; mean 6 SD shown.

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A 10-min incubation of 107 Jurkat cells with 100 mM14C-CALP1 resulted in substantial accumulation of theeptide inside these cells (approximately 2,500 cpm,hich is the equivalent of 566 ng, Fig. 2). In fact, since

hese cells have a diameter of approximately 10 mm,his value approximates equimolar concentrations be-ween the cells and the extracellular bath solution,xcluding an active transport mechanism for this up-ake. Furthermore, 30 min after the loading of theseells, there was minimal leakage if they were kept at°C, but substantial leakage if they were at 37°C (Fig.). The finding that the efflux of the peptide after thiseriod of time was not equal to the influx suggestshere is a sequestration.

If the only mechanism by which the peptide is enter-ng the cells is by passive diffusion, we would expect ainear dose response curve. However, when we testedhis, we found a non-linear dose-response (Fig. 3). Thisuggests that the peptide is either being taken up byhe cells, at least in part, by some mediated transportechanism or there is a sequestration of the peptide

nside the cell as suggested above (Fig. 2). The timeourse for the uptake of the peptide indicates that by0 min loading had reached a plateau (Fig. 4). Conse-

FIG. 5. HPLC analysis of 14C-CALP1 recovered from the cytoso-ic fraction of Jurkat T lymphocytes. (A) HPLC chromatograph ofnlabelled CALP1. (B–E) Fractions taken every minute wereounted for associated radioactivity. (B) 14C-CALP1 control resus-ended in H2O prior to injection. Note, approximately 1 min timeelay due to solution movement between the HPLC lamp and frac-ionator. (C) 14C-CALP1 control resuspended in cytosolic fraction ofurkat T cells prior to injection. (D) Cytosolic fraction extracted fromurkat T cells previously loaded with 14C-CALP1 for 10 min at 37°C,mmediately transferred to 4°C, where they were acid stripped andhen washed three times. (E) Cytosolic fraction of unlabeled Jurkat

cells run on HPLC.

466

xperiments.Subcellular fractionation of Jurkat cells loaded with

he 14C-CALP1 showed that, like CaM (13), it is presentn fractions representing nuclei, cytoplasm, and mem-rane (data not shown). HPLC analysis done on cyto-olic fractions recovered from Jurkat cells incubatedith 14C-CALP1 for 10 min, indicates that most of theeptide is still intact (Fig. 5). The retention time for theeptide recovered from these cells (Fig. 5D) matcheshe retention time for unlabeled CALP1 (Fig. 5A), 14C-ALP1 (Fig. 5B), and 14C-CALP1 added to cytosolicxtract from normal Jurkat cells (Fig. 5C). The smalleak at the beginning of Fig. 5D indicates some degra-ation (,5% of the total product).Two approaches were taken to establish an interac-

ion of 14C-CALP1 with CaM within the interior of theells. Near neighbor analysis was conducted on Jurkatells pre-incubated with 14C-CALP1 for 10 min, follow-ng which the cells were washed, and treated with theonspecific crosslinking reagent DSS. This method re-ults in the crosslinking between free amino groupshat are in close proximity (14, 15). After crosslinking,he cells were disrupted and analyzed by Western blot-ing with a monoclonal antibody specific for CaM (Fig.A) (12). The gel was then also subjected to autoradiog-

FIG. 6. 14C-CALP1 associates with a band recognized by an:CaM antibody on Western blot of Jurkat T lymphocytes loadedith peptide and crosslinked with DSS. Lanes (1–3) represent cell

ysate control, cell lysate from crosslinked cells, and cells incubatedith 14C-CALP1 and crosslinked, respectively. For this, 107 cells per

ondition were incubated with 100 mM 14C-CALP1 at 37°C for 10 minnd then immediately transferred to 4°C, where they were acidtripped and then washed three times. These cells were then incu-ated with the crosslinking agent DSS for 1 h, before being lysed andubjected to SDS–PAGE (10 mg total protein each condition). Thisas then blotted using a monoclonal-a:CaM antibody (A) then used

o expose radiographic film (B). Black arrow indicates a new bandecognized by a:CaM antibody after crosslinking with associatedadioactivity when cells were incubated with 14C-CALP1. Gray ar-ows indicate radioactivity associated with crosslinked 14C-CALP1 atower MW.

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aphy (Fig. 6B). From this figure it can be seen thatrosslinking cells with DSS (lane 2) results in a bandorresponding to CaM as well as an extra band at higholecular weight (MW) that is recognized by anti:CaM

ntibody (black arrow). We assume that the high mo-ecular weight complex corresponds to CaM bound tots target proteins. Interestingly, when cells were in-ubated with 14C-CALP1 and then crosslinked, thereas radioactivity associated with the high moleculareight complex (black arrow) but not with free CaM.rom this result, it is tempting to speculate that asoon as CALP binds CaM, the complex immediatelynd strongly interacts with one or more target protein.n this scenario, the CALP bound to free CaM repre-ents such a small percentage of the total that it iselow the resolution of the system. There are also twoands of radioactivity at lower MW (grey arrows),hich most likely represent peptide crosslinked to it-

elf or some other small molecule(s).In a second approach to confirm an association ofALP and CaM, we took advantage of a Jurkat cell linehich has been stably transfected with a Tet-off CaM

ense gene. Under normal conditions these cells over-xpress CaM, and the addition of Tet reduces the ex-

FIG. 7. Correlation of 14C-CALP1 associated in bound fractiony tet-off CaM expressing Jurkat cells. Tet-off CaM expressing Juoncentrations of tet, which Western blot analysis reveals that as cere loaded with 100 mM 14C-CALP1 at 37°C for 10 min and then

hen washed three times. Cells were then lysed and subjected to imoat IgG. (B) The bound fraction was then counted and expressednd expressed as mean 6 SD.

467

ression in a dose-dependent manner (Fig. 7A). Thus,ells grown in various concentrations of Tet were incu-ated with 14C-CALP1, washed, and then crosslinkedith DSS. Whole cell lysates were immunoprecipitatedith either polyclonal goat anti:CaM antibody or nor-al goat IgG and protein A/G agarose beads. This

mmunoprecipitate was then washed, and the radioac-ivity associated with the bound versus the unboundraction was determined for each sample. As Fig. 7Bhows, there is a strong correlation of bound peptideith the concentration of CaM; however, there wasegligible radioactivity associated with the sampleshich either had not been crosslinked or with those

hat had been immunoprecipitated with normal goat IgG.The present studies test the hypotheses that a pep-

ide could be designed de novo and targeted to anntracellular protein. In order to study this, we initiallyetermined whether the peptide is able to enter theell, whether it remains intact inside the cell, andnally whether it is able to interact with its target,aM. In order to do this we studied a radiolabeledersion of CALP1 being taken up by Jurkat T cells.Due to the size and hydrophobicity of the peptide, we

ypothesized that it would be able to passively diffuse

a:CaM immunoprecipitate, with amount of CaM being expressedat cells (107 per condition) were incubated with several differenting a dose dependent decrease in CaM expression (A). These cellsmediately transferred to 4°C, where they were acid stripped andnoprecipitation with either polyclonal a:CaM antibody or normala percentage of total cpm. Each condition was done in triplicate

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ACKNOWLEDGMENTS

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he dose-response curve, as well as the inequality be-ween the influx and efflux of the peptide, indicatedhat diffusion may not be the only mechanism involvedn the uptake. Either some active transport mecha-ism, such as carrier mediated transport or more likelysequestration of the peptide in the interior of the cell,ay account for this. HPLC analysis demonstrated

hat the peptide stayed intact at least for 10 min aftereing taken up by the cell. There was a small degree ofegradation, which would probably increase over time;owever, the resilience of the peptide was surprising.his may be due to an interaction of the peptide withroteins in the interior of the cell, which protect it fromeptidase activity, or a sequestration of the peptidento compartments of the cell which are not susceptibleo such activity. The ability of the peptide to enter theucleus could be due to the permeability of the peptidehroughout the cell or some active transport into theucleus. Indeed, it is tempting to speculate that it isue to an activation of CaM by CALP, since Ca21-ctivated CaM has been shown to translocate into theucleus (16).The crosslinking studies demonstrate an interaction

f CALP with CaM, albeit in an indirect manner. DSSs a nonspecific crosslinker; however, it is useful forear neighbor analysis, since it binds free aminoroups which are within a few angstroms distance fromne another. The results of the immunoprecipitationxperiments indicating a correlation of the amount ofeptide pulled down with the amount of CaM beingxpressed by the cell further supports the hypothesishat CALP is interacting with CaM in the interior ofhe cell. These results reinforce our earlier findings of

specific interaction between CALP and CaM andemonstrate the ability of this interaction to occur initu. Since CaM plays such a prominent signaling rolen cells, a specific and productive interaction by CALPould result in useful biological consequences. For ex-mple, CaM acts as a Ca21 sensor for several types ofon channels, regulating their activity based on thentracellular Ca21 concentration (17–28). In fact, weave found that CALP is an effective blocker of the-methyl-D-aspartate receptor (NMDA-R) channel, asell as a non-selective cation channel, present in Jur-at T cells, which is activated by HIV-1 gp120 (11).urthermore, this block was dependent upon CaM forhe NMDA-R. Interestingly blocking these channelsith CALP resulted in an inhibition of apoptosis me-iated by a Ca21. Given the important role CaM playsithin a cell, the utility of the CALP peptides for the

tudy and impedance of Ca21-mediated signaling path-ays may be far reaching. For example, given thatALP targets intracellular CaM, it may be possible to

ouple other substances to these peptides, in essencehuttling this complex to CaM and therefore CaM-inding proteins.

468

Thanks to Diane Weigent for editorial assistance in the prepara-ion of this manuscript. This work was supported by grants to J.E.B.rom the National Institutes of Health (MH52527, AI37670, andS29719) and by grants to J.M.M. from the National Institutes ofealth (CA72823 and AR43225) and by a Veterans Administrationerit Review.

EFERENCES

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2. Baranyi, L., Campbell, W., Ohshima, K., Fujimoto, S., Boros, M.,and Okada, H. (1995) The antisense homology box: A new motifwithin proteins that encodes biologically active peptides. NatureMedicine 1, 894–901.

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Cellular Uptake and in Situ Binding of a Peptide Agonist for Calmodulin