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...

Abstract:The application of non-thermal atmospheric pressure plasma raises a hope for the new wound healing strategies. Next to its antibacterial effect it is known to stimulate skin cells. However, monocytes are also needed for the complex process of a wound healing. This study investigates the impact of plasma on the intracellular signaling events in the primary human monocytes. The proliferative MEK-ERK (MAPK/ERK kinase-extracellular signal-regulated kinase) pathway was activated by short plasma treatment times. In contrast, an induction of the apoptotic JNK (c-Jun N-terminal kinase) cascade as well as activation of caspase 3 were observed after long plasma exposure. These findings indicate that monocytes can be differentially stimulated by plasma treatment and may contribute to the proper wound recovery.

Chlamydiae are bacterial pathogens that grow in vacuolar inclusions. Dendritic cells (DCs) disintegrate these compartments, thereby eliminating the microbes, through auto/xenophagy, which also promotes chlamydial antigen presentation via MHC I. Here, we show that TNF-α controls this pathway by driving cytosolic phospholipase (cPLA)2-mediated arachidonic acid (AA) production. AA then impairs mitochondrial function, which disturbs the development and integrity of these energy-dependent parasitic inclusions, while a simultaneous metabolic switch towards aerobic glycolysis promotes DC survival. Tubulin deacetylase/autophagy regulator HDAC6 associates with disintegrated inclusions, thereby further disrupting their subcellular localisation and stability. Bacterial remnants are decorated with defective mitochondria, mito-aggresomal structures, and components of the ubiquitin/autophagy machinery before they are degraded via mito-xenophagy. The mechanism depends on cytoprotective HSP25/27, the E3 ubiquitin ligase Parkin and HDAC6 and promotes chlamydial antigen generation for presentation on MHC I. We propose that this novel mito-xenophagic pathway linking innate and adaptive immunity is critical for effective DC-mediated anti-bacterial resistance.