Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

Abstract

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.

Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

“…Thus, as seen in Figure 3 , the stimulation above the basal is much less for kidney cortex (graph C), a tissue rich in many types of A-GPCRs, than for CHO cell Y2 expression (graph D), and the activation by rabbit piriform cortexY2 receptors is overwhelmed by contributions of other receptors that produces a very large basal 90 kDa binding of [ 35 S ]GγS (graph E). CHO cell expressions of the human Y1 (graph B) and especially of the human Y4 receptor (graph F) appear to have more extensive activation than CHO-Y2 expression (graph D), which could relate to the large acidic clusters of the Y2 receptor (see [ 52 ]).…”

Half-Life Extending Modifications of Peptide YY3–36 Direct Receptor-Mediated Internalization

“…Instead, we hypothesize that the C18acid may restrict PYY 3−36stimulated Y 2 R internalization dynamics by interactions with the extracellular matrix. 58,68 Anionic side chains in Y 2 R's Nterminal extracellular segment PDPEPE, located adjacent to the PYY 3−36 binding pocket, have been shown as a primary factor for the reduced internalization rate of Y 2 R compared to the Y 1 receptor. 58,68 The mechanism behind the PDPEPE segment's control of Y 2 R internalization rates is unknown, but association between the domain and connector proteins of the extracellular matrix has been suggested to play a role.…”

Drug Delivery Systems, CNS Protection, and the Blood Brain Barrier

“…But cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies [ 39 ]. Contrary to this, unmasking by permeabilizing but nondetaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO phenylarsine oxide [ 40 ]. However, depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide.…”

“…However, depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. It shows the presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells [ 40 ]. However, in spite of their potential, many existing peptide and protein drugs (P/P drugs) are rendered ineffective in the treatment because of their inability to deliver and sustainability within the brain.…”