Ligand-induced Internalization and Recycling of the Human Neuropeptide Y2 Receptor Is Regulated by Its Carboxyl-terminal Tail

Abstract

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...

Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins

“…In contrast, nonvisual arrestins show only a ϳ3-5-fold binding differential (14 -16, 19) and were reported to bind unphosphorylated receptors in response to their activation (42,(63)(64)(65)(66)(67)(68). Thus, it appears that non-phosphate binding elements likely contribute more significantly to the binding of non-visual arrestins to their cognate receptors.…”

“…Therefore, the observed changes in spin label mobility can be directly attributed to the effects of receptor binding. The spin labels attached to arrestin-2 at positions 68,70,71,73,167,191,234,238, and 246 become substantially immobilized upon binding to dark P-Rh. Strong immobilization of these surface residues suggests that the binding of P-Rh causes the residues to come into direct contact with either the receptor itself, the adjacent membrane, or with other arrestin elements.…”

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

“…Direct binding studies with arrestin-2 and arrestin-3 revealed that both nonvisual subtypes bind with fairly high affinity to inactive phosphorylated GPCRs, with this binding reaching 70 -80% of maximum achieved with corresponding active phosphoreceptors (60 -62). Therefore, to determine whether agonist-independent phosphorylation of GPCRs by GRK5 has any functional consequences, we examined arrestin recruitment in intact cells, using BRET between ␤2AR tagged with Renilla luciferase at the C terminus and arrestins tagged with Venus at the N terminus (35,36,40,63,64). The BRET ratio in these experiments was determined as described previously (35,36,63), but because we were primarily interested in GRK-dependent increase of arrestin recruitment, we calculated net BRET as an increase in BRET ratio in the presence of exogenous GRK, as compared with cells that only express endogenous GRKs along with tagged ␤2AR and arrestins.…”

Silent Scaffolds

“…To establish a quantitative in vivo arrestin-3-JNK3 interaction assay, we included the same forms of arrestin-3 N-terminal-tagged with Venus, which worked very well in the BRET-based arrestinreceptor interaction assay (18,19,25,40,41) (Fig. 1).…”