Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB₁ cannabinoid receptor

Highlights

• CB₁R binds β-arrestin2 but not β-arrestin1 upon activation.

• Agonist-induced internalization of CB₁R is β-arrestin2 dependent.

• Constitutive CB₁R internalization does not require β-arrestin2.

• Agonist-induced and constitutive CB₁R endocytosis are distinctly regulated processes.

Abstract

CB₁ cannabinoid receptor (CB₁R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of β-arrestins in the regulation of CB₁R function are not completely understood. In this study, we followed CB₁R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB₁R binds β-arrestin2 (β-arr2), but not β-arrestin1. Furthermore, both the expression of dominant-negative β-arr2 (β-arr2-V54D) and siRNA-mediated knock-down of β-arr2 impaired the agonist-induced internalization of CB₁R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB₁R. However, both constitutive and agonist-induced internalization of CB₁R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB₁R, β-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB₁R are different.

Introduction

Internalization of G protein-coupled receptors (GPCRs) is an important process in the regulation of receptor function. Although its main function is the modulation of receptor number on the cell surface, thereby adjusting the sensitivity of the cell to external stimuli, it also plays role in the resensitization and signaling of GPCRs (Ferguson, 2001, Hunyady and Catt, 2006, Shenoy and Lefkowitz, 2011). At the molecular level, β-arrestins are key regulatory proteins of receptor internalization, as they can bind to activated GPCRs, as well as to clathrin and the adaptor protein AP-2, thus directing the receptor towards clathrin-mediated endocytosis (Shenoy and Lefkowitz, 2011). β-Arrestins also mediate receptor desensitization, as their binding to the activated GPCRs causes the uncoupling of the receptor from its cognate G protein (Shenoy and Lefkowitz, 2011). Furthermore, they play important roles in the activation of G protein-independent signal transduction pathways, e.g. the activation of MAP kinases, phosphatidylinositol 3 kinase, Akt or the small GTP-ase RhoA (DeWire et al., 2007, Wei et al., 2003).

β-Arrestin1 (β-arr1) and β-arrestin2 (β-arr2) are two ubiquitously expressed isoforms of β-arrestins (Ferguson, 2001). Although β-arrestin binding is a general property of most activated GPCRs, the selectivity and stability of these binding shows receptor specific differences. Namely, class A receptors (e.g. the β₂ adrenergic receptor) bind β-arr2 with a higher affinity than β-arr1, and this binding is transient, i.e. it can only be detected at or near the plasma membrane. In contrast, class B GPCRs, such as the AT₁ angiotensin receptor, bind both β-arr1 and β-arr2 with relatively high affinity and form stable complexes with β-arrestins, so that β-arrestins remain bound to the receptor after internalization and can be detected on intracellular vesicles (Oakley et al., 2000).

The CB₁ cannabinoid receptor (CB₁R) belongs to the superfamily of G protein-coupled receptors (GPCRs). The receptor plays role in many important physiological processes, such as learning, thinking, nociception or the regulation of food-intake (Pacher et al., 2006). Activation of presynaptic CB₁Rs by postsynaptic endocannabinoid release, which mediates retrograde transmission, is a key regulatory mechanism in the central nervous system, but paracrine activation of CB₁Rs with a similar mechanism can also occur in extraneural tissues (Freund et al., 2003, Gyombolai et al., 2012, Sanchez et al., 2001, Turu et al., 2009, Szekeres et al., 2012). The cellular signaling events following CB₁R activation are mainly associated with the activation of heterotrimeric G_i/o-proteins and include inhibition of adenylyl cyclases, activation of K_ir channels, inhibition of Ca_v channels and phosphorylation and activation of different subtypes of mitogen-activated protein kinases (MAP kinases) (Turu and Hunyady, 2010). G protein-independent signaling events following CB₁R stimulation have also been reported (Sanchez et al., 2001).

Similar to most GPCRs, CB₁R internalizes upon agonist stimulation. This has been demonstrated in many cell lines, including CHO (Rinaldi-Carmona et al., 1998), AtT20 (Hsieh et al., 1999, Jin et al., 1999, Roche et al., 1999), F11 (Coutts et al., 2001), neuroblastoma N18TG2 (Keren and Sarne, 2003) and HEK293 (Keren and Sarne, 2003, Leterrier et al., 2004) cells, as well as in hippocampal neurons, which naturally express CB₁R (Coutts et al., 2001, Leterrier et al., 2006). According to different studies, this agonist-induced CB₁R endocytosis occurs via clathrin- and/or caveolin-mediated pathways in different cell types (Bari et al., 2008, Hsieh et al., 1999, Keren and Sarne, 2003, Wu et al., 2008).

Numerous studies suggest that β-arr2 is involved in the regulation of CB₁R. Co-expression of both GRK3 and β-arr2 was needed for the proper desensitization of the receptor in Xenopus oocyte (Jin et al., 1999), and dominant negative GRK2 and β-arrestin constructs reduced CB₁R desensitization in hippocampal neurons (Kouznetsova et al., 2002). In β-arr2 knockout mice desensitization and downregulation of CB₁R were impaired in certain regions of the central nervous system (Nguyen et al., 2012). Recruitment of β-arr2 to the activated CB₁R has also been demonstrated (Daigle et al., 2008a). Mutation of amino acids S426 and S430 was shown to inhibit receptor desensitization as well as late phase receptor endocytosis, but not β-arrestin binding (Daigle et al., 2008b, Jin et al., 1999). It has been demonstrated that serine and threonine residues at the C-terminus of CB₁R are involved in its β-arr2 binding and agonist-induced endocytosis (Daigle et al., 2008b, Hsieh et al., 1999). In contrast to the various studies that clearly point to an interaction between CB₁R and β-arr2 upon agonist stimulation, no direct data have been hitherto presented concerning the β-arr1 binding of CB₁R. In some structural studies, the association of β-arr1 with a synthesized CB₁R C-terminus has been shown (Bakshi et al., 2007, Singh et al., 2011), however, such binding has not been demonstrated with the intact CB₁R in living cells.

Constitutive internalization of CB₁R (i.e. spontaneous internalization in the absence of CB₁R agonists) has also been detected in hippocampal neurons, as well as in CHO and HEK cells (Leterrier et al., 2004, Leterrier et al., 2006, McDonald et al., 2007a, Turu et al., 2007). It has been suggested that constitutive CB₁R internalization is the consequence of its basal activity, since inverse agonist treatment or inhibition of basal activity with a DAG lipase inhibitor (e.g. tetrahydrolipstatin) interfered with this process (Leterrier et al., 2004, Leterrier et al., 2006, Rinaldi-Carmona et al., 1998, Turu et al., 2007). However, other studies have concluded that constitutive internalization occurs independently of receptor activity (McDonald et al., 2007a, McDonald et al., 2007b, Kleyer et al., 2012). The latter statement raises the possibility that constitutive and agonist-induced internalization of CB₁R may occur via distinct endocytic mechanisms (McDonald et al., 2007a). However, no evidence has been hitherto presented showing that these two processes are truly different in that they require distinct endocytic machinery to take place.

Our main goal was, considering the important consequences of β-arrestin recruitment in the regulation of cell function, to characterize the β-arrestin binding properties of CB₁R and to reveal possible differences in constitutive and agonist-driven CB₁R endocytosis by investigating the role of β-arrestins in these processes.