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Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor‐specific shuttle system represents a promising solution. [F7,P34]‐NPY analogues were modified by solid‐phase peptide synthesis, enabling non‐covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co‐internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT‐PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide‐based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor‐specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects. [ABSTRACT FROM AUTHOR]

Peptides have beneficial properties for therapeutic applications due to their excellent target specificity, high affinity and activity, low toxicity and predictable metabolism. Thus, they became an important tool for research and medical purpose. However, peptides are fragile molecules, therefore, in order to be used as therapeutic agents they need to be stabilised by non-peptidic modifications. Required improvements of these peptide properties include longer half-life, higher bioavailability, increased potency and efficiency. Strategies to achieve these goals have been identified in the last years, with synthetic strategies to obtain sufficient amounts becoming increasingly important. In this review, peptides are discussed with respect to their therapeutic applications. Examples from glucagon-like peptide 1 receptor agonists are shown. Peptides are compared to small molecules and antibodies with respect to advantages and disadvantages in therapeutic applications. [ABSTRACT FROM AUTHOR]

Copyright of Nachrichten aus der Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential. [ABSTRACT FROM AUTHOR]

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N-terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y2 receptor on living cells. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract: The export of newly synthesized proteins, including G protein-coupled receptors (GPCR), from the endoplasmic reticulum (ER) and further transport to the plasma membrane is a tightly regulated process. ER export and subsequent cell surface targeting of GPCR is initially mediated through COPII-coated vesicles. It is governed by specific amino acid sequences located in extracellular as well as intracellular receptor domains, for example in the C-terminus (CT) of the receptor. Herein, we determined the role of the CT in the anterograde transport of the human neuropeptide Y receptor (hYR) type 2. We identified a short sequence motif in the membrane proximal CT: Y(x)3F(x)3F in the region of the putative 8th helix has a critical functional relevance for the anterograde transport of hY2R, since its deletion leads to accumulation of the receptor in the ER. It is sequence and position specific. Furthermore we identified a distinct role of C-terminal sequences in hY1R, hY2R, hY4R and hY5R. Regulation of hY5R export is regulated by a different mechanism as compared to hY2R. Different sequence elements with respect to function and localization are involved as demonstrated by the construction of a hY2/hY5 receptor chimera and a noneffective deletion in the region of helix eight in the hY5R. In contrast to hY2R, deletion of the corresponding helical segment F(x)3L(x)3F has no influence on anterograde transport of hY1R, whereas deletion of F(x)3I(x)3V in hY4R restrains the receptor to the Golgi apparatus. Interestingly this pattern is not mirrored by repression of COPII vesicle transport by Sar1[H79G] overexpression. Whereas the 8th helix is involved before or at the level of Sar1 dependent export pathways in the ER for the hY2R, in hY4R helix eight is involved at later stages of anterograde transport. [Copyright &y& Elsevier]

The article discusses the current knowledge on neuropeptide Y receptor (YRs) functions, their involvement in stringent human diseases and how studies opened opportunities to develop new drug candidates in clinical application for diagnostics and therapy. It points out that the neuropeptide Y hormone family is made up of three closely related peptides namely NPY, PYY and PP. It indicates the importance of the receptor internalization process which represents a key step in cancer treatment although therapeutic compounds for clinical use is still lacking.

SK-N-MC neuroepithelioma cells are routinely cultured and widely used as a model system in biochemical and pharmacological experiments. To clarify the gene expression patterns of SK-N-MC cells with respect to G protein-coupled receptors and signaling network components, we describe in this report the transcription profile of the cell line. Following the traditional pathway from genome to proteome, selected examples are further examined at the level of protein expression and by functional assays. cRNA targets derived from total RNA extracts were hybridized to Affymetrix Human Genome U133A GeneChip arrays, and the data were analyzed and grouped according to functional aspects. Results obtained for neuropeptide Y (NPY) Y1, Y5, and orexin Ox1 receptors were confirmed by RT-PCR. It is surprising that we found the presence of both NPY receptor subtypes and the absence of the orexin receptor at the mRNA level. Receptor-binding experiments confirmed NPY binding of the Y1 receptor in the nanomolar range but gave no evidence for high expression levels of Y5 receptor subtypes on the cell surface. Protein expression was assayed with immunoblots by using antibodies directed against selected Gα protein subunits. The presence of at least Gαs, Gαi3, and Gαi2 subunits was indicated. [ABSTRACT FROM AUTHOR]

After nerve lesion, a biphasic upregulation of nerve growth factor synthesis occurs in nonneuronal cells. Two fundamentally different regulatory principles underlie this phenomenon. A previously described tissue-extrinsic mechanism depends on macrophages invading the lesioned nerve and their secreted products, such as interleukin 1. It is responsible for the second delayed response. Here we demonstrate a novel mechanism of lesion-induced NGF regulation, which makes use exclusively of tissue-intrinsic elements. Sciatic nerve contains a potent preformed NGF-inducing activity. It is released within minutes after nerve lesion and is responsible for the first rapid NGF increase, which occurs within hours after injury. This type of regulatory mechanism may allow for matching of NGF synthesis with the severity of the lesion. [Copyright &y& Elsevier]

AdipoR1 and AdipoR2 are newly discovered members of the huge family of seven-transmembrane receptors, but both receptors are structurally and functionally different from G-protein-coupled receptors. Little is known about the oligomerization of the AdipoRs. Here, we show the presence of endogenous AdipoR1 dimers in various cell lines and human muscle tissue. To directly follow and localize the dimerization, we applied bimolecular fluorescence complementation (BiFC) in combination with flow cytometry. We visualized and quantified AdipoR1 homodimers in HEK293 cells. Moreover, we identified a GxxxG dimerization motif in the fifth transmembrane domain of the AdipoR1. By mutating both glycine residues to phenylalanine or glutamic acid, we were able to modulate the dimerization of AdipoR1, implicating a role for the GxxxG motif in AdipoR1 dimerization. Furthermore, we tested whether the AdipoR1 ligand adiponectin had any influence on receptor dimerization. Interestingly, we found that adiponectin decreases the receptor dimerization in a concentration-dependent manner. This effect is mainly mediated by segments of the collagen-like domain of full-length adiponectin. Accordingly, this is the first direct read-out signal of adiponectin at the AdipoR1 receptor, which revealed the involvement of specific amino acids of both the receptor and the ligand to modulate the quaternary structure of the AdipoR1. [ABSTRACT FROM AUTHOR]

The article reports the structures of the human Y1, Y2, and Y4 receptors in complex neuropeptide (NPY) or pancreatic polypeptide (PP). Topics discussed include distinct binding poses of the peptide upon coupling to differing receptors, conformational plasticity of the peptide in recognizing the NPY receptors, and NPY signal recognition and transduction.

GPCR internalization, which is induced by arrestin recruitment, is an important mechanism for the regulation of signaling and receptor quantity at the cell surface. In this study, differences in the mechanism of arrestin-3 (arr-3) recruitment to the neuropeptide Y 1 and Y 2 receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The Y 1 R displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the Y 2 R has a lower affinity for arr-3. Internalization is induced by arrestin binding, but arr-3 is released from the receptor and remains at the membrane while the receptor internalizes. Moreover, the deletion of the finger loop region of arr-3 reduces its agonist-dependent recruitment to the Y 2 R significantly, but not to the Y 1 R suggesting different binding conformations. For the first time, the formation of a supercomplex consisting of Y receptor, Gα 0 protein and arrestin was studied by BRET-assay. We demonstrated that the Y 1 R is able to bind Gα 0 protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the Y 2 R no supercomplex formation was observed. By substituting the C-terminus or specific residues within the intracellular loop 1 and 2 of the receptors, the arr-3 recruitment of the Y 1 R and Y 2 R can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα 0 protein and arrestin in response to Y 1 versus Y 2 receptor activation and identified the molecular determinants. [ABSTRACT FROM AUTHOR]

The human neuropeptide Y 4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa , we aimed to investigate the molecular mechanism of hY 4 R desensitization and endocytosis. The role of distinct segments of the hY 4 R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY 4 R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif 7.78 EESE HLPL ST VH T EV S KG S 7.96 was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y 4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis. [ABSTRACT FROM AUTHOR]

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 Y2 receptor (hY2R), 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 enzyme-linked 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 373DSXTEXT379 was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif 347DXXXSEXSXT356 promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs (357FKAKKNLEVRKN368), which serves as an inhibitory element. We found that hY2R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY2R 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 hY2R, which is likely applicable to other GPCRs. [ABSTRACT FROM AUTHOR]

Abstract: Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin. [Copyright &y& Elsevier]

Abstract: Agonist stimulation of G-protein coupled receptors (GPCRs) results in the redistribution of the receptor from the cell surface into intracellular compartments through the process of endocytosis. Monitoring ligand-mediated internalization of GPCRs in living cells has become experimentally accessible by applying fluorescent reagents and fluorescence microscopy. By using cell lines that transiently, stably or endogenously express the human Y receptor (hYR) subtypes hY1R, hY2R, hY4R and hY5R and differently fluorescently tagged receptor proteins we were able to unravel further details concerning the internalization behavior of this multi-receptor/multi-ligand system. For the first time we could show that also the hY2R is internalized with a rate which is comparable to the hY1R and the hY4R. In contrast, the hY5R was internalized much slower and the rate remained unaffected by co-expression with other hYR subtypes. Furthermore receptor subtype co-expressing cells and selectively binding peptides revealed a receptor subtype selective internalization. By using novel hY5/hY2 receptor chimera the receptor subtype dependent differences in hY receptor internalization could be identified on a molecular level. [Copyright &y& Elsevier]

Y receptors (YRs) are G protein-coupled receptors whose Y1R, Y2R, and Y5R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y4Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp6.59 to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y2R and Y5R, Arg33 is the binding partner of Asp6.59, in Y1R and Y4R, Arg35 of human PP and NPY interacts with Asp6.59. Furthermore, Arg25 of PP and NPY is involved in ligand binding only at Y2R and Y5R. This suggests significant differences in the docking of YR ligands between Y1/4R and Y2/5R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs. [ABSTRACT FROM AUTHOR]