**2. Structural divergence of the cannabinoid receptors from class A GPCRs**

The CB1 receptor is a class A (Rhodopsin-like) GPCR (**Figure 1**). Different phylogenetic studies and multidimensional scaling analysis of Class A GPCRs classify cannabinoid receptors (CB1/CB2) into one cluster along with the endothelial differentiation G-protein coupled receptors (EDGRs) (including Sphingosine 1-phosphate receptors (S1P) and Lysophosphatidic acid receptors (LPA)) [9–12]. Receptors from those families, except for the LPA4–6, share common sequence divergence from other Class A GPCRs. Specifically, the absence of helix kinking proline residues in TMH2 and TMH5, and the absence of a disulfide bridge between the EC-2 loop and C3.25 at the EC end of TMH3. Instead, they share an internal disulfide bridge in the EC-2 loop, a conserved PxxGW motif at the EC end of TMH4, in addition to a Y5.39 that forms an aromatic pi-pi stack with W4.64 in that motif resulting in a similar shape of the EC2 loop as seen in the crystal structures for the CB1, S1P1 , and LPA1 receptors [6, 7, 13, 14]. At the

binding site, they share a common basic residue (K/R 3.28) on TMH3 and an aromatic residue (F/Y 2.57) on TMH2. In addition, the S1P receptors are like CB1/CB2 in the presence of E1.49 at TMH1. E1.49 has been reported to be a key interaction site for pregnenolone (an endogenous negative allosteric modulator that protects the brain from *cannabis* intoxication) with CB1 [15], while the LPA1–3 receptors share a W5.43 with CB1/CB2 that has been shown to affect

**Figure 1.** Helix net representation of the hCB1 receptor. The most highly conserved residue in each helix is shown in bold. Residues are numbered using the BW#: Ballesteros-Weinstein residue numbering system in GPCRs which uses the X.YY format; X denotes the transmembrane helix number and (YY) denotes residue position relative to the most

receptors recognize lipid-derived ligands that have been shown to bind to the receptor by diffusing from bulk lipid towards the binding site via a transmembrane portal [6, 7, 14, 17, 18].

, and the cannabinoid

Structural Insights from Recent CB1 X-Ray Crystal Structures

http://dx.doi.org/10.5772/intechopen.80783

35

antagonist binding to the cannabinoid receptors [16]. In addition, S1P1

conserved residue in the helix (X.50). Loop regions are numbered using absolute sequence numbers.

GPCRs have a common architecture of seven transmembrane helices (TMHs) joined by extracellular (EC) and intracellular (IC) loops of varied lengths, in addition to an extracellularly extending N terminus, and an intracellular C terminus that begins with an amphipathic alpha helical segment (Helix 8) oriented parallel to the cell membrane. In Class A GPCRs, the binding site for the endogenous ligand is generally formed by the EC core within the TMH bundle, and may extend to EC loops, referred to as the orthosteric binding site. Ligands may also bind

Due to the various physiological functions mediated by GPCRs, they are considered major targets for drug discovery and design of novel therapeutics. However, understanding the structure-function relationship of these proteins and the design of high affinity, selective ligands that target these receptors requires a detailed knowledge of the three-dimensional structure of the receptor in general and of the ligand binding site in specific. However, structural characterization for membrane proteins in general has been a challenge due to their low expression in recombinant hosts and their inherent instability in surfactants. It was not until the year 2000 that the first high resolution GPCR structure was resolved by X-ray crystallography, Rhodopsin in its inactive state [1]. The following 10 years witnessed the release of other inactive state crystal structures of class A GPCRs (e.g. the Adenosine A2A, and the β1 and β2 adrenergic receptors [2–4]), in addition to the release of the active state crystal structure of Rhodopsin in complex with a synthetic peptide resembling the C-terminus of the G-alpha subunit of transducing [5]. Available structures during that time served as templates for homology modeling for other GPCRs including the CB1 receptor. And parallel with biophysical studies, available crystal structures provided structural insights for their activation mechanism. A breakthrough in GPCR structural characterization has been achieved in the last 8 years with more than 200 structures for different GPCRs being deposited in the Protein Data Bank, including the CB1 inactive and active state crystal structures which have been resolved in 2016, and in 2017 respectively [6–8]. Before that, structural characterization of CB1 orthosteric as well as allosteric binding domains have been extensively studied via mutations, site-directed labeling, mass spectrometry, SAR studies, and in-silico methods, and will be

to distinct (allosteric) binding sites in the receptor.

34 Recent Advances in Cannabinoid Research

discussed in detail throughout this chapter.

loop as seen in the crystal structures for the CB1, S1P1

**A GPCRs**

**2. Structural divergence of the cannabinoid receptors from class** 

The CB1 receptor is a class A (Rhodopsin-like) GPCR (**Figure 1**). Different phylogenetic studies and multidimensional scaling analysis of Class A GPCRs classify cannabinoid receptors (CB1/CB2) into one cluster along with the endothelial differentiation G-protein coupled receptors (EDGRs) (including Sphingosine 1-phosphate receptors (S1P) and Lysophosphatidic acid receptors (LPA)) [9–12]. Receptors from those families, except for the LPA4–6, share common sequence divergence from other Class A GPCRs. Specifically, the absence of helix kinking proline residues in TMH2 and TMH5, and the absence of a disulfide bridge between the EC-2 loop and C3.25 at the EC end of TMH3. Instead, they share an internal disulfide bridge in the EC-2 loop, a conserved PxxGW motif at the EC end of TMH4, in addition to a Y5.39 that forms an aromatic pi-pi stack with W4.64 in that motif resulting in a similar shape of the EC2

, and LPA1

receptors [6, 7, 13, 14]. At the

**Figure 1.** Helix net representation of the hCB1 receptor. The most highly conserved residue in each helix is shown in bold. Residues are numbered using the BW#: Ballesteros-Weinstein residue numbering system in GPCRs which uses the X.YY format; X denotes the transmembrane helix number and (YY) denotes residue position relative to the most conserved residue in the helix (X.50). Loop regions are numbered using absolute sequence numbers.

binding site, they share a common basic residue (K/R 3.28) on TMH3 and an aromatic residue (F/Y 2.57) on TMH2. In addition, the S1P receptors are like CB1/CB2 in the presence of E1.49 at TMH1. E1.49 has been reported to be a key interaction site for pregnenolone (an endogenous negative allosteric modulator that protects the brain from *cannabis* intoxication) with CB1 [15], while the LPA1–3 receptors share a W5.43 with CB1/CB2 that has been shown to affect antagonist binding to the cannabinoid receptors [16]. In addition, S1P1 , and the cannabinoid receptors recognize lipid-derived ligands that have been shown to bind to the receptor by diffusing from bulk lipid towards the binding site via a transmembrane portal [6, 7, 14, 17, 18].
