Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor protolymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24^q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/ Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion. ß

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulation in all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.Keywords: oligonucleotide-peptide conjugates; cellular uptake; cell-penetrating peptides.The effectiveness of antisense oligonucleotides and peptide nucleic acids in modifying mammalian cell function can be improved substantially by covalent attachment or complexing with natural cell-penetrating peptide sequences [1][2][3][4]. This increase in biological activity has been commonly attributed to an enhanced cellular uptake of the conjugates [5][6][7]. The peptide components used to date have been protein-derived sequences that exhibit very different structural properties, ranging from lipophilic to unstructured and highly positively charged sequences [5,[7][8][9][10][11] as well as to strongly structured amphipathic ones [12][13][14][15]. The structural requirements for the peptide moiety and the necessity for covalent attachment remain controversial.In the present study we investigated the influence of the complexing or covalent tagging of phosphorothioate oligonucleotides with cationic model peptides of different structure forming properties (Table 1, Fig. 1) upon the cellular uptake. The a-helical amphipathic 18-mer model peptide used here (I) and its derivatives (exhibiting reduced helicity or amphipathicity) were previously shown to possess analogous cell penetrating properties to the above mentioned natural sequences [16][17][18]. We observed extensive cellular uptake of naked oligonucleotides as well as of their peptide derivatives. The uptake rates were all within an order of magnitude for a given cell type and oligonucleotide length irrespective of the mode of peptide binding or peptide structural properties. Conjugation or complexing of the oligonucleotides with the most widely used natural vector peptide, derived from the homeodomain of Antennapedia [19], led to comparable results. Our results therefore imply other aspects than an improved translocation across mammalian plasma membranes such as increased affinity to target structures or interactions with oligonucleotide bind...

SUMMARYNorleucine''-substance P and 4-chlorophenylalanine 7;8-Norleucine 11 -. .substance P were synthesized by fragment condensation. Phel H/7*8-norleuc 7 r i e l l -substance P was obtained by catalytic tritiation of the precursor geptide with a specific radioactivity of 27 Cilmole and full biological activity.Recently we reported the tritium labelling of the modified 3 * partial sequence of substance P, acetyl-Lys-Phel HI-Phe-Gly-Leu-NleNH2, by catalytic tritiation of the corresponding synthetic Cpa-containing precursor peptide 111. The labelled peptide was purified using preparative paper electrophoresis and possessed a specific radioactivity of 23 Ci/mmole 111 -a surprisingly high labelling rate for the tritiation of a 4-chlorophenylalanine peptide. The labelled compound had high biological activity in vivo but in receptor studies no specific binding

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