Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

Calcitonin-derived carrier peptides

The recent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.     

Calcitonin-derived peptide carriers: mechanisms and application

Among the family of the so-called cell-penetrating peptides (CPP) sequences derived from the native peptide hormone human calcitonin (hCT) have recently proven to translocate different bioactive molecules across cellular membranes. Herein, we give an extensive summary of the development of hCT-derived carrier peptides, beginning with the therapeutic nasal administration of full-length hCT. Hence, N-terminally truncated hCT fragments were investigated and subsequently optimised to extend their field of application. The latest generation of hCT-derived carrier peptides are highly effective, branched peptides. The current state of the art is reviewed concerning the structural requirements, mechanistic assumptions and metabolic features of these peptides as well as experiments proving their excellent carrier potential.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Metabolic Cleavage and Translocation Efficiency of Selected Cell Penetrating Peptides: A Comparative Study with Epithelial Cell Cultures

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Palpha] and [Pbeta], when in contact with either subconfluent HeLa, confluent MDCK or Calu-3 epithelial cell cultures. Additionally, through analysis of their cellular translocation efficiency, we evaluated possible relations between metabolic stability and translocation efficiency. Metabolic degradation kinetics and resulting metabolites were assessed using RP-HPLC and MALDI-TOF mass spectrometry. Translocation efficiencies were determined using fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Between HeLa, MDCK and Calu-3 we found the levels of proteolytic activities to be highly variable. However, for each peptide, the individual degradation patterns were quite similar. The metabolic stability of the investigated CPPs was in the order of CF-SAP = CF-hCT(9-32)-br > [Pbeta]-IAF > [Palpha] and we identified specific cleavage sites for each of the four peptides. Throughout, we observed higher translocation efficiencies into HeLa cells as compared to MDCK and Calu-3, corresponding to the lower state of differentiation of HeLa cell cultures. No direct relation between metabolic stability and translocation efficiency was found, indicating that metabolic stability in general is not a main limiting factor for efficient cellular translocation. Nevertheless, translocation of individual CPPs may be improved by structural modifications aiming at increased metabolic stability.     

Fatty acyl moieties: improving Pro‐rich peptide uptake inside HeLa cells

Abstract: In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro‐rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro‐rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.     

Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec)

Abstract

Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (P_app) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.     

Maurocalcine as a Non Toxic Drug Carrier Overcomes Doxorubicin Resistance in the Cancer Cell Line MDA-MB 231

Purpose  The aim of this study is to overcome tumour cell resistance that generally develops after administration of commonly used anti-cancer drugs, such as doxorubicin. Methods  Recently, cell penetrating peptides have been used for their ability to deliver non-permeant compounds into cells. One such cell penetrating peptide, maurocalcine, has been isolated from the venom of a Tunisian scorpion. Herein, we report the effects of doxorubicin covalently coupled to an analogue of maurocalcine on drug-sensitive or drug-resistant cell lines MCF7 and MDA-MB 231. Results  We demonstrated the in vitro anti-tumoral efficacy of the doxorubicin maurocalcine conjugate. On a doxorubicin-sensitive cancer cell line, the maurocalcine-conjugated form appears slightly less efficient than doxorubicin itself. On the contrary, on a doxorubicin-resistant cancer cell line, doxorubicin coupling allows to overcome the drug resistance. This strategy can be generalized to other cell penetrating peptides since Tat and penetratin show similar effects. Conclusion  We conclude that coupling anti-tumoral drugs to cell penetrating peptides represent a valuable strategy to overcome drug resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antimicrobial and Cytolytic Activities and Plausible Mode of Bactericidal Action of the Cell Penetrating Peptide Penetratin and Its Lys‐linked Two‐Stranded Peptide

The cell penetrating peptide, penetratin (RQIKIWFQNRRMKWKK‐NH₂) showed potent antimicrobial activity (MIC: 0.5–4 μm ) without any cytotoxicity against mammalian cells. This study investigated the effect of linking together two peptide chains of penetratin on antimicrobial and cytolytic activities and plausible mode of bactericidal action. Two‐stranded penetratin was prepared by a simultaneous solid‐phase synthesis of the two strands of a single lysine residue attached to the solid support. Two‐stranded penetratin markedly increased cytolytic activity against human erythrocytes and NIH‐3T3 mouse fibroblast cells without a significant effect on antimicrobial activity. This finding suggested that penetratin is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Circular dichroism analysis revealed that the α‐helical content of the membrane‐bound penetratin was unaffected by two‐stranded Lys‐linkage. Penetratin had very weak ability in the depolarization of membrane potential of intact Staphylococcus aureus cells and the fluorescent dye leakage of calcein‐entrapped negatively charged bacterial membrane‐mimicking vesicles. In contrast, two‐stranded penetratin significantly caused membrane depolarization and dye leakage. These results suggest that the two‐stranded penetratin induces a significant change in its mode of bactericidal action from the intracellular‐target mechanism to the membrane‐targeting mechanism.     

Drug discovery by formulation design and innovative drug delivery systems (DDS)

This review describes studies on drug discovery using a rational formulation design and innovative, drug delivery systems (DDS) for biomaterials such as therapeutic peptides and nucleotides. The microcapsules of the LH-RH superagonist leuprorelin acetate prepared using the new in-water drying method and biodegradable polymers, such as PLGA and PLA, could achieve a long-term sustained release for 1-6 months thereby facilitating easily treatment of hormone-dependent diseases, prostate cancer, endometriosis, and precocious puberty. This DDS technology showed an improvement in patient QOL and highly promoted the clinical value of the agonist. Moreover, PLGA microcapsules of siRNAs against VEGF, cFLIP, Raf-1, and Int6 have also been developed to treat various cancers and arteriosclerosis obliterans. To develop therapeutic nucleotides, a particle design is created using functional peptides, such as cell penetrating peptides (CPP), nuclear localizing signals (NLS), tight junction reversible openers (AT1002), bombesin, and dynein light chain-associated sequences. siRNA use should lead to a paradigm shift in drug discovery against various diseases. Tat analog with NLS could enhance the potency of a vaginal DNA vaccine. The artificial Tat CPP of STR-CH(2)R(4)H(2)C synthesized in our laboratory could efficiently deliver siRNAs into many types of cells and enhance the therapeutic effects for treating sarcoma, atopic dermatitis, allergic rhinitis, and asthma by intratumor injection and inhalation of the nanoparticles. Tat and AT1002 analogs used to treat atopic dermatitis in mice increased cell membrane permeability to siRelA, a siRNA against a subclass of NF-κB, and exhibited striking therapeutic and preventive effects.     

Cationic Amphiphilic Polyproline Helices: Side‐Chain Variations and Cell‐Specific Internalization

Cell‐penetrating peptides present an attractive and efficient tool for the delivery of a variety of cell impermeable cargoes across the cellular membrane. Cell‐penetrating peptides usually consist of short basic peptide sequences that are internalized by a variety of cell lines. Most cell‐penetrating peptides lack cell specificity, however, which greatly limits their use as efficient therapeutic agents. Herein, we present two cell‐penetrating peptides displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups. The uptake efficiency of these cationic amphiphilic polyproline helices was studied in seven different cell lines, six cancerous (MCF‐7, HOS, HT1080, HeLa, KB‐FD, KB3‐1) and one non‐cancerous (WI 38). The cationic amphiphilic polyproline helix P11LRR at 50 μm showed high specificity toward MCF‐7 breast cancer cells. Co‐culture experiments with P11LRR demonstrated almost exclusive internalization by MCF‐7 cells and not WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across some cell lines. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell‐penetrating peptides can strongly influence the extent and specificity of cellular internalization.     

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.     

The vimentin-tubulin binding site peptide (Vim-TBS.58-81) crosses the plasma membrane and enters the nuclei of human glioma cells

Cell-penetrating peptides (CPPs) can translocate through the plasma membrane and localize in different cell compartments providing a promising delivery system for peptides, proteins, nucleic acids, and other products. Here we describe features of a novel cell-penetrating peptide derived from the intermediate filament protein vimentin, called Vim-TBS.58-81. We show that it enters cells from a glioblastoma line via endocytosis where it distributes throughout the cytoplasm and nucleus. Moreover, when coupled to the pro-apoptogenic peptide P10, it localizes to the nucleus inhibiting cell proliferation. Thus, the Vim-TBS.58-81 peptide represents an effective vector for delivery of peptides and potentially a broad range of cargos to the nucleus.     

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5?µM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.     

On the Cellular Uptake and Membrane Effect of the Multifunctional Peptide,TatLK15

Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:293–304, 2014     

The role of anion exchange in the uptake of Pb by human erythrocytes and Madin-Darby canine kidney cells

Anion exchange (AE) plays a critical role in regulating intracellular pH in erythrocytes and epithelial cells and has been suggested to facilitate the transport of lead (Pb) across the erythrocyte cell membrane. In this study we examined the role of AE in the uptake of Pb by human erythrocytes and by Madin-Darby canine kidney (MDCK) cells, the kidney epithelial cell line. Functional AE in MDCK cells was evidenced by: increased uptake of SO(4)(2-) at pH 6.0 over pH 7.0, and inhibition of SO(4)(2-) uptake by the AE inhibitor 4, 4'-diisothiocyanostilbene-2, 2'- disulfonic acid (DIDS) as well as by non-halide anions. Accumulation of Pb into MDCK cells was time and temperature dependent. DIDS inhibited uptake of Pb into human erythrocytes but not MDCK cells. In conclusion, uptake of Pb into erythrocytes but not kidney epithelial cells occurs through AE.     

Can the Cellular Internalization of Cargo Proteins Be Enhanced by Fusing a Tat Peptide in the Center of Proteins? A Fluorescence Study

The aim of this study was to investigate whether the cellular uptake of cargo proteins can be enhanced by fusing a Tat peptide in the center of proteins; glutathione-S-transferase (GST)-Tat-green fluorescent protein (GFP) and GST-GFP-Tat proteins were first constructed and expressed. The cellular internalization of both proteins was then evaluated and compared in HeLa cells using fluorescent microscopy and flow cytometry, as well as the transdermal delivery in human skin using confocal microscopy. Results from in vitro cell experiments showed that GST-Tat-GFP protein efficiently internalized into HeLa cells when a Tat peptide was fused in the center of proteins, whereas its efficiency is lower than that of GST-GFP-Tat protein with a Tat peptide terminal fused. Ex vivo transdermal delivery data also demonstrated that the lower efficiency of GST-Tat-GFP penetrating through human stratum corneum layer when compared with GST-GFP-Tat. Furthermore, both GST-GFP-Tat and GST-Tat-GFP presented a various degree of a mixture of cytoplasmic diffuse staining and punctate surface staining, and the pattern of distribution varied considerably in HeLa cell experiments depending on the concentration of protein used. Therefore, an improved mechanism for Tat-conjugated proteins was proposed, in which Tat-conjugated proteins were first associated with cell membrane, then accumulated on the cell surface, and finally internalized into cells by pore formation mechanism.     

Insights into the uptake mechanism of NrTP, a cell-penetrating peptide preferentially targeting the nucleolus of tumour cells

Nucleolar targeting peptides are 14-15 residue-long sequences designed by structural minimization of a snake toxin (J Med Chem 2008;50:7041). Peptides such as NrTP1 (YKQCHKKGGKK GSG) and analogues are capable of penetrating human cervix epithelial carcinoma cells and homing into their nucleoli. We now show that NrTP1 similarly penetrates and localizes in the nucleolus of tumour cells derived from human pancreatic (BxPC-3) and human ductal mammary gland (BT-474) carcinomas. Live cell confocal microscopy imaging, combined with flow cytometry analysis of cells arrested to defined phases of their cycle, confirms that NrTP1 uptake and nucleolar homing are independent of cell cycle phase. Peptide uptake is significantly reduced at low temperature. Also, drugs inhibiting chlatrin-mediated endocytosis severely decrease uptake, pointing to a clathrin-dependent route as the primary NrTP1 internalization mechanism. These results highlight nucleolar targeting peptides not only as a novel and efficient class of cell-penetrating peptides but also for their exceptional ability to target preferentially an essential and dynamic subnuclear structure such as the nucleolus.     

A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.