A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

Abstract

The cannabinoid receptors belong to the class A (rhodopsin family) of G protein-coupled receptors (GPCRs).2 The second cannabinoid receptor subtype, CB2 (2), is highly expressed throughout the immune system (3, 4) and has been described in the central nervous system under both pathological (5) and physiological conditions (6). All known CB2 ligands are highly lipophilic. In fact, the CB2 endogenous cannabinoid, sn-2-arachidonoylglycerol (2-AG) (7,8), is synthesized on demand from the lipid bilayer itself in a two-step process in which phospholipase C-␤ hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol, which is then hydrolyzed by diacylglycerol lipase to yield 2-AG (9, 10). After 2-AG interaction with the membraneembedded CB receptor, it is hydrolyzed to arachidonic acid and glycerol by a membrane-associated enzyme, monoacylglycerol lipase (11). As revealed by the crystal structures of rhodopsin (12-15), the ␤ 2 -adrenergic receptor (AR) (16 -18), ␤ 1 -AR (19), and adenosine A2A receptor (20), the general topology of a GPCR includes the following: 1) an extracellular (EC) N terminus; 2) seven transmembrane ␣-helices (TMHs) arranged to form a closed bundle; 3) loops connecting TMHs that extend intra-and extracellularly; and 4) an intracellular (IC) C terminus that begins with a short helical segment (helix 8) oriented parallel to the membrane surface. Agonists bind inside the crevice formed by the TMH bundle and produce conformational changes on the IC face of the receptor that uncover previously masked G protein-binding sites (21), which then lead to G protein coupling. Biophysical studies using a variety of techniques indicate that ligand-induced receptor activation produces the following changes: 1) a conformational change in the W6.48 "toggle switch" within the ligand binding pocket (22); 2) a change in the relative orientations of TMH3 and -6 that breaks an IC "ionic lock" (23-28), with the intracellular end of TMH6 moving away from TMH3 by hinging and moving up toward lipid (27); 3) the uptake of two protons (29); and 4) an influx of water (30).Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, AM841, enters the CB2 receptor via the lipid bilayer (1). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. We report here microsecond time scale unbiased

Cell membranes… and how long drugs may exert beneficial pharmacological activity in vivo

“…Indeed, targets at the plasma membrane lose part of their natural microenvironment when cells are broken apart [87] [95][96][97], biophysical and computational approaches, such as X-ray scattering, nuclear magnetic resonance and in silico molecular dynamics, offer promise to herald a much clearer picture of…”

“…The cause of this discrepancy remains elusive. However, as illustrated by recent advances in our understanding of cannabinoid, opsin and sphingosine 1-phosphate receptor interactions [95][96][97], biophysical and computational approaches, such as X-ray scattering, nuclear magnetic resonance and in silico molecular dynamics, offer promise to herald a much clearer picture of Figure 4 Experimental in vitro and in vivo manifestations of drug 'rebinding'. (A, B) Simulated time-wise decline in in vitro target occupancy by a fast-(panel A, dissociation half-life (t 1/2 ) = 20 min) and a candesartan-like slower-dissociating radioligand (panel B, dissociation t 1/2 = 120 min) under washout conditions.…”

“…Examples are provided for ligand-G-protein-coupled receptor tandems. An approach based on two-dimensional diffusion within the membrane has also been proposed to take place with several other ligand-receptor tandems [35,[95][96][97]99] 'microkinetic' model provides an elegant explanation for these observations. Drug partitioning may sometimes interfere with the correct interpretation of experimental observations and, to address this eventuality, it may be useful to compare the results from different experimental approaches.…”

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction

“…CB 2 has also been reported to play an important role in central immune responses during neuropathic pain in mice (16). We have previously performed microseconds long MD simulations of the CB 2 endogenous ligand, sn-2-arachidonoylglycerol (2-AG), entering and activating CB 2 via the lipid bilayer (17). Activation of CB 2 has been shown experimentally to produce coupling to G␣ i inhibitory protein (18 -20).…”

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

“…2). It seems likely that other lipid ligands may enter via similar routes, and indeed that has also been suggested for the cannabinoid receptor family, although this has not yet been confirmed by a structure (43).…”