Constitutive Activation Drives Compartment-Selective Endocytosis and Axonal Targeting of Type 1 Cannabinoid Receptors

Abstract

The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.

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

“…This is in line with the data published by McDonald et al, which showed no alterations in the cellular distribution of CB 1 R upon inverse agonist treatment in neurons (McDonald et al, 2007a). In contrast, a previous study using inverse agonist treatment (Leterrier et al, 2006), and our study using DAGL-inhibitor treatment (Turu et al, 2007) have shown an inhibition of basal endocytosis of the receptor upon these treatments, suggesting a role for basal CB 1 R activity in this process. Since active receptors internalize by β-arr2-dependent mechanism, those results may seem to be contradictory with our present results, where we now find that interfering with β-arr2 expression or function does not have effect on constitutive endocytosis.…”

“…However, in hippocampal neurons, it has been demonstrated that agonist treatment leads to internalization (Coutts et al, 2001). These data, together with differences detected in the regulation of constitutive internalization (Leterrier et al, 2006;McDonalds et al, 2007a) are in good agreement with our results…”

“…This has been demonstrated in many cell lines, including CHO (Rinaldi-Carmona et al, 1998), AtT20 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 CB 1 R (Coutts et al, 2001;Leterrier et al, 2006). According to different studies, this agonist-induced CB 1 R 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).…”

“…spontaneous internalization in the absence of CB 1 R agonists) has also been detected in hippocampal neurons, as well as in CHO and HEK cells (Leterrier et al, 2004(Leterrier et al, , 2006McDonald et al, 2007a;Turu et al, 2007). It has been suggested that constitutive CB 1 R internalization is the consequence of its basal activity, since inverse agonist treatment or inhibition of basal activity with a DAG lipase inhibitor (e.g.…”

“…It has been suggested that constitutive CB 1 R 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, , 2006Rinaldi-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, , 2007bKleyer et al, 2012).…”