A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48 -60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48 -60). These included such peptides as the D-amino acid-and arginine-substituted Tat-(48 -60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4°C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg) n (n ؍ 4 -16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n ϳ 8) for the efficient translocation.Recently, methods have been developed for the delivery of exogenous proteins into living cells with the help of membranepermeable carrier peptides such as HIV-1 1 Tat-(48 -60) and Antennapedia-(43-58) (1-11). By genetically or chemically hybridizing these carrier peptides, the efficient intracellular delivery of various oligopeptides and proteins was achieved. One of the most amazing examples is the Tat--galactosidase fusion protein (4), which has a molecular mass as high as 120 kDa.Intraperitoneal injection of the protein resulted in delivery of the protein with -galactosidase activity to various tissues in mice, including the brain. The peptide-mediated approaches would allow the incorporation of peptides containing unnatural amino acids or nonpeptide molecules such as fluorescence probes. These methods would become powerful tools not only for therapeutic purposes as an alternative to gene delivery, but also for the understanding of the mechanisms behind fundamental cellular events, such as signal transduction and gene transcription.Besides the potential of Tat-(48 -60) as a protein carrier, the internalization mechanism of the peptide attracted our interest. For example, Tat-(48 -60) (GRKKRRQRRRPPQ) is a highly basic and hydrophilic peptide, which contains 6 arginine and 2 lysine residues in its 13 amino acid residues. However, the peptide was reported to be translocated through the cell membranes in 5 min at a concentration of 0.1 M (2). Internalization of the peptide was not inhibited even at 4°C. The peptide is less toxic to cells than other basic membrane-interacting agents. The above features suggested that the internalization mechanism of Tat-...
Membrane-permeable arginine-rich peptides, such as HIV-1 Tat-(48-60), HIV-1 Rev-(34-50), and flock house virus (FHV) coat-(35-49), have been shown to possess the ability to transfect COS-7 cells with luciferase-coding plasmid as efficiently as polyarginine (MW 5000-15 000) and polylysine (MW 9800). Not only these virus-derived cationic peptides but also oligoarginines of 4-16 residues were found to be able to transfect cells. In the case of the Tat, FHV, and octaarginine peptides, N-terminal stearylation of the peptides increases the transfection efficiency by approximately 100 times to reach the same order of magnitude as that of LipofectAMINE, one of the most efficient commercially available transfection agents. Also, a certain correlation was observed between the transfection efficiency of stearyl-(Arg)n peptides (stearyl-Rn: n = 4, 8, 12, 16) and the membrane permeability of the corresponding (Arg)n peptides (Rn).
Aberrant zinc (Zn) homeostasis is associated with abnormal control of mammalian growth, although the molecular mechanisms of Zn's roles in regulating systemic growth remain to be clarified. Here we report that the cell membrane-localized Zn transporter SLC39A14 controls G-protein coupled receptor (GPCR)-mediated signaling. Mice lacking Slc39a14 (Slc39a14-KO mice) exhibit growth retardation and impaired gluconeogenesis, which are attributable to disrupted GPCR signaling in the growth plate, pituitary gland, and liver. The decreased signaling is a consequence of the reduced basal level of cyclic adenosine monophosphate (cAMP) caused by increased phosphodiesterase (PDE) activity in Slc39a14-KO cells. We conclude that SLC39A14 facilitates GPCR-mediated cAMP-CREB signaling by suppressing the basal PDE activity, and that this is one mechanism for Zn's involvement in systemic growth processes. Our data highlight SLC39A14 as an important novel player in GPCR-mediated signaling. In addition, the Slc39a14-KO mice may be useful for studying the GPCR-associated regulation of mammalian systemic growth.
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