Phosducin, β-arrestin and opioid receptor migration

https://doi.org/10.1016/S0014-2999(99)00223-XGet rights and content

Abstract

Internalization of G protein-coupled opioid receptors depends on multiple criteria, including the affinity of drugs to their receptors and the state of the receptor–G protein interaction. Most recent studies reveal that cytosolic components like phosducin and arrestin interfere with receptor internalization, that is phosducin impairs receptor phosphorylation and arrestin enhances endocytosis by uncoupling the receptor from its G protein. This study was designed to examine the mutual effect phosducin and arrestin exert on receptor endocytosis. Neuronal NG 108-15 hybrid cells transiently expressing the μ-opioid receptor, which has been fused to green fluorescence protein, were employed to study internalization of the fluorescent μ-opioid receptor construct in living cells by means of confocal laser scanning microscopy. Fluorescent μ-opioid receptors were detected in drug-naive cells both at the cell membrane and at cell surface protrusions, most likely filopodia, microspikes and retraction fibres. The opioid receptors present in the cell membrane internalize upon etorphine (1 nM) exposure, a process clearly blocked in cells overexpressing phosducin. However, coexpression of both phosducin and β-arrestin 1 reverses this blockade. In contrast to etorphine, morphine fails to internalize μ-receptors expressed in NG 108-15 cells. When arrestin is overexpressed in these cells, morphine gains the ability to induce endocytosis, and this process is left unaffected by phosducin. The findings suggest that endocytosis of activated μ-opioid receptors primarily depends on arrestin-triggered uncoupling of the receptor from its G protein complex. Drug-induced receptor phosphorylation appears of subordinate significance for receptor internalization.

Introduction

Activation of G protein-coupled receptors, including the opioid receptors, brings about desensitization and possibly receptor internalization (Lefkowitz, 1998). The underlying biochemical processes have been reported to depend on multiple mechanisms, such as the affinity of ligands and their intrinsic activity (Kovoor et al., 1998), the phosphorylation of receptors (Zhang et al., 1998), the activity of arrestins (Krupnick and Benovic, 1998), and the formation of clathrin-coated pits (Lin et al., 1997). On the other hand, there is evidence to demonstrate that sequestration of receptors does not require each of the criteria mentioned, e.g., receptor phosphorylation (Murray et al., 1998), and no strict relation exists between receptor affinity of drugs and their ability to cause receptor internalization (Gaudriault et al., 1997). The present study was designed to more closely investigate opioid receptor endocytosis under the influence of the cytosolic components phosducin (Lee et al., 1987) and β-arrestin 1 (Krupnick and Benovic, 1998).

The phosphoprotein phosducin (Lee et al., 1992) has been reported to represent an ubiquitous cytosolic component (Danner and Lohse, 1996), which upon receptor stimulation translocates towards the cell membrane (Schulz et al., 1998a). Phosducin binds to Gβγ liberated from the G protein trimer by receptor activation (Blüml et al., 1997), thereby preventing G protein-coupled receptor kinases (GRKs) to bind to the occupied G protein dimer. Gβγ serves cytosolic GRKs as membrane anchor (Hawes et al., 1994), a prerequisite to phosphorylate agonist-occupied receptors. Phosphorylation of receptors, including the opioid receptors, is required to induce desensitization (Hawes et al., 1994; Pitcher et al., 1995; Schulz et al., 1998b). Moreover, binding of phosducin to Gβγ hinders reassociation of G protein subunits to form a trimer (Lee et al., 1992), which eventually results in an increased GTPase activity of Gα subunits (Bauer et al., 1992). These functions of phosducin, specifically those relating to neutralization of Gβγ, may contribute to inhibition of receptor-mediated endocytosis (Lin et al., 1997), a mechanism most recently documented for opioid receptors in NG 108-15 neuronal hybrid cells stably overexpressing phosducin (Schulz et al., 1999).

Further cytosolic components most critical for the process of receptor endocytosis are the arrestins (Lefkowitz, 1998). These proteins redistribute following receptor activation and tightly bind to the C-terminal of phosphorylated receptors, thereby uncoupling the receptor from its G protein (Ferguson et al., 1996). As consequence of the interruption of signal transmission endocytosis of receptors will be initiated (Krupnick and Benovic, 1998). These findings seem to attribute also to μ-opioid receptors, as β-arrestin represents a strong cellular constituent accounting for rapid receptor endocytosis in cooperation with G protein-coupled receptor kinases (Kovoor et al., 1997).

The studies reported here examine the effect of phosducin and β-arrestin on internalization of μ-opioid receptor activated by etorphine and morphine, respectively. These ligands were selected since etorphine is well known to trigger rapid endocytosis of μ-receptors, while morphine has been communicated to lack the ability to bring about receptor internalization (Keith et al., 1996). Employing these opioids we tested the mutual effect of phosducin, which is known to prevent internalization (Schulz et al., 1999), and β-arrestin, which stimulates internalization (Kovoor et al., 1997). These cellular processes were made visible in living NG 108-15 neuronal hybrid cells, using confocal laser scanning microscopy to follow the migration of μ-opioid receptors fused to green fluorescence protein (Schulz et al., 1999).

Section snippets

Cell culture

Neuroblastoma×glioma hybrid NG 108-15 cells were raised in DMEM medium supplied with 10% fetal calf serum (Schulz et al., 1998a) and penicillin (100 IU/ml)/streptomycin (100 μg/ml). If not otherwise mentioned, experiments were conducted with cells at 70% confluency.

Compounds

Morphine–HCl was purchased from Merck (Mannheim, Germany), etorphine–HCl was provided by National Institute of Drug Abuse (USA). Phalloidin and all further compounds were obtained from Sigma (Taufkirchen, Germany).

Antibodies

The β-arrestin

Distribution of μ-opioid receptors (fluorescence) in NG 108-15 cells

Confocal microscopy of a single optical section from NG 108-15 cells expressing the μ-opioid receptor/EGFP fusion protein revealed fluorescence almost exclusively associated with the cell membrane (Schulz et al., 1999). This study examines the location of fluorescence (EGFP-tagged μ-receptors) throughout cells by monitoring confocal sections at 1 μm intervals from bottom to top. Fig. 1 (images A to J) displays the distribution of fluorescence of a representative NG 108-15 cell transfected to

Discussion

μ-Opioid receptors fused to EGFP are incorporated into the membrane of NG 108-15 cells, they retain their function and will be sequestered upon exposure to the appropriate opioid (Schulz et al., 1999). Here we report that fluorescence (μ-opioid/EGFP constructs) associated with the cell membrane exhibits a characteristic distribution. That is, confocal microscopy of optical sections close to the bottom of the cell reveals morphological structures resembling pseudopodia or nerve growth cones (

References (30)

Cited by (0)

View full text