No abstract

We developed multifunctional fluorescent nanoparticles suitable for the nonviral delivery of negatively charged molecules like RNA. Therefore, we incorporated the recently developed branched hCT-derived carrier peptide hCT(18-32)-k7 on the surface of luminescent quantum dots (QDs). Besides detailed characterization of our QD-peptide conjugates concerning stability, toxicity, and uptake mechanism. we used them for efficient RNA delivery into different cell lines. The results of our studies indicate the involvement of more than one endocytotic uptake pathway in the internalization process. Furthermore, we could show that the QD-peptide bioconjugates exhibit no effect on cell viability and possess high stability inside living cells. The efficacy of our newly designed constructs for oligonucleotide drug delivery is highlighted by the successful intracellular transport of Cy-3 labeled RNA. Moreover, by using the chemotherapeutic chloroquine the efficient release of the assemblies out of endosomes was demonstrated. These results prove that our multifunctional platforms are versatile tools for diagnostic and therapeutic imaging purposes applicable for biologically active siRNA or aptamer sequences.

Agonist-induced internalization of G protein-coupled receptors plays an important role in signal regulation. The underlying mechanisms of the internalization of the human neuropeptide Y 2 receptor (hY 2 R), as well as its desensitization, endocytosis, and resensitization are mainly unknown. In the present study we have investigated the role of carboxyl-terminal (C-terminal) Ser/Thr residues and acidic amino acids in regulating receptor internalization, arrestin interaction, and recycling by fluorescence microscopy, cell surface enzymelinked immunosorbent assay, and bioluminescence resonance energy transfer in several cell lines. Strikingly, C-terminal truncation mutants revealed two different internalization motifs. Whereas a distal motif 373 DSXTEXT 379 was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif 347 DXXXSEX-SXT 356 promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs ( 357 FKAKKNLEVRKN 368 ), which serves as an inhibitory element. We found that hY 2 R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY 2 R C terminus is involved in multiple molecular events that regulate internalization, interaction with arrestin-3, and receptor resensitization. Our findings provide novel insights into complex mechanisms of controlled internalization of hY 2 R, which is likely applicable to other GPCRs. G protein-coupled receptors (GPCRs)2 constitute the largest family of cell-surface receptors with ϳ800 known human subtypes. Members of this family share a common architecture of seven membrane-spanning ␣-helices connected by extra-and intracellular loops. GPCRs can be activated by a wide variety of extracellular stimuli and regulate diverse physiological processes (1). Because they are responsible for a multitude of cellular responses and their dysfunction can result in many diseases (2-4), this receptor family represents highly important pharmaceutical targets. More than one-third of the currently available therapeutics act on GPCRs (1). Whereas antagonists mainly prevent receptor interactions with endogenous ligands, agonist drugs might lead to distinct effects in addition to receptor activation, e.g. functional antagonism by receptor removal from the surface (5), direct intracellular cross-signaling (6), and may behave as biased ligands (7). Current information about the complex intracellular network of GPCRs regulation is rather limited. Therefore, it is of great interest to unravel the mechanisms and regulation modalities of receptor internalization and subsequent resensitization processes.The Y 2 R is one of four human neuropeptide Y (NPY) receptor subtypes (hY 1 R, hY 2 R, hY 4 R, and hY 5 R) and belongs to the rhodopsin-like superfamily (class A) of GPCRs. YRs together with their three native ligands, NPY, pancreatic polypeptide (PP), and p...

Background: CRFR1 regulates the physiological response to stress and is implicated in the manifestation of depression. Results: SAP97 interacts and co-localizes with CRFR1, suppresses CRFR1 endocytosis, and is required for CRFR1-mediated ERK1/2 phosphorylation. Conclusion: SAP97 functionally regulates CRFR1 trafficking and signaling. Significance: This is the first documentation of functional regulation of CRFR1 by a specific PDZ protein.

NPY, PYY and PP constitute the so-called NPY hormone family, which exert its biological functions in humans through YRs (Y₁, Y₂, Y₄ and Y₅). Systematic modulation of YR function became important as this multireceptor/multiligand system is known to mediate various essential physiological key functions and is involved in a variety of major human diseases such as epilepsy, obesity and cancer. As several YRs have been found to be overexpressed on different types of malignant tumors they emerge as promising target in modern drug development. Here, we summarize the current understanding of YRs function and the molecular mechanisms of ligand binding and trafficking. We further address recent advances in YR-based drug design, the development of promising future drug candidates and novel approaches in YR-targeted tumor diagnostics and therapy opportunities.