Parathyroid hormone-related protein (PTHrP) contains a nuclear localization signal (NLS) sequence within 87-107. NLS sequences are generally capable of penetrating cellular membranes due to a richness of basic amino acid residues, and thus have been used as cell-penetrating peptides (CPPs) to translocate biologically active peptides/proteins into cells. The NLS sequence of PTHrP is not exception to this finding; however, PTHrP(87-107) contains 2 acidic glutamate residues at 99 and 101 within the basic amino acid stretch, which is not commonly observed in other CPPs such as HIV-1 Tat(48-60). In this study, we indicated structure-function relationship of the PTHrP NLS to understand the effect of acidic glutamate residues on cell permeability and intracellular localization. We chemically synthesized PTHrP(87-107) and its N-terminally truncated analogues. Their intracellular localization pattern was analyzed by microscopy, radioimmunoassay, and fluorescence-activated cell sorting. Although all analogues were translocated into cells, internalization by the cytoplasm and/or nucleus was length-dependent; specifically, PTHrP(97-107), PTHrP(95-107), and PTHrP(93-107) were more frequently localized in the cytoplasm. We assume that reduction in the net positive charge within PTHrP NLS analogues resulted in increased cytoplasm-translocation activity. We propose that PTHrP(97-107) is a useful carrier peptide for delivery and expression of cargo molecules in the cytoplasm.Peptide-mediated protein translocation is a popular method of delivering biologically active peptides/ proteins into cells. This transport system is conducted by short peptides (< 30 amino acids) that translocate large molecules into cells [reviewed in (14)]. These so-called cell-penetrating peptides (CPPs) are found in protein transduction domains or nuclear localization signals (NLSs) of viral proteins (5). CPPs have been applied to in vitro biological and clinical studies (6). For instance, short peptides from HIV-1 Tat protein including Tat(48-60) and Tat(47-57) have been widely used to deliver cargo molecules into cells (26,29). The amino acid sequences of CPPs are rich in basic amino acid residues. Interestingly, a short peptide containing only 8 arginine residues was translocatable (7). Parathyroid hormone-related protein (PTHrP) was identified as a tumor-derived secretory protein (17). The N-terminal 20 residues of the 141-amino acid peptide showed structural similarity to parathyroid
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