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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 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 (373)DSXTEXT(379) was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif (347)DXXXSEXSXT(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.

SUBMITTER: Walther C

PROVIDER: S-EPMC3009885 | biostudies-other | 2010 Dec

REPOSITORIES: biostudies-other

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Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail.

Walther Cornelia C Nagel Stefanie S Gimenez Luis E LE Mörl Karin K Gurevich Vsevolod V VV Beck-Sickinger Annette G AG

The Journal of biological chemistry 20101018 53

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 flu ...[more]

PMID: 20959467

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Project description:G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

Dataset Information

Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail.

Publications

Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail.

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