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The brain cannabinoid (CB1) receptor that mediates numerous physiological processes in response to marijuana and other psychoactive compounds is a G protein coupled receptor (GPCR) and shares common structural features with many rhodopsin class GPCRs. For the rational development of therapeutic agents targeting the CB1 receptor, understanding of the ligand-specific CB1 receptor interactions responsible for unique G protein signals is crucial. For a more than a decade, a combination of mutagenesis and computational modeling approaches has been successfully employed to study the ligand-specific CB1 receptor interactions. In this review, after a brief discussion about recent advances in understanding of some structural and functional features of GPCRs commonly applicable to the CB1 receptor, the CB1 receptor functional residues reported from mutational studies are divided into three different types, ligand binding (B), receptor stabilization (S) and receptor activation (A) residues, to delineate the nature of the binding pockets of anandamide, CP55940, WIN55212-2 and SR141716A and to describe the molecular events of the ligand-specific CB1 receptor activation from ligand binding to G protein signaling. Taken these CB1 receptor functional residues, some of which are unique to the CB1 receptor, together with the biophysical knowledge accumulated for the GPCR active state, it is possible to propose the early stages of the CB1 receptor activation process that not only provide some insights into understanding molecular mechanisms of receptor activation but also are applicable for identifying new therapeutic agents by applying the validated structure-based approaches, such as virtual high throughput screening (HTS) and fragment-based approach (FBA).
G-protein coupled receptors (GPCRs), composed of seven transmembrane (TM) spanning helices (H1-H7) interconnected by three intracellular loops (I1-I3) and three extracellular loops (E1-E3) [1,2], are known to be among the most important drug targets [3,4] (for reviews, see [5-7]). For the rational development of therapeutic agents targeting a specific GPCR, it is crucial to understand the ligand-receptor interactions that determine the effectiveness of the ligand for modulating receptor activity toward unique G protein signals. If the ligand binding affinity is defined by how strongly a ligand binds to a receptor and the receptor efficacy is defined by how efficiently a receptor activates the coupled G protein, a ligand as a drug candidate should exhibit high binding affinity and more importantly desirable binding efficacy. Thus, it should be at the center of the rational development of GPCR drugs to understand the ligand-initiated receptor conformational change responsible for GPCR signaling [8,9].
Understanding of binding efficacy in GPCRs is challenging due to the conformational complexity of GPCRs [10,11]. Even in the absence of ligands GPCRs exhibit basal activity, suggesting that GPCRs are in motion with inherent conformational flexibility. Conformational equilibrium of a GPCR between the inactive state and the active state can be modified by ligand binding . By definition, agonists activate the receptor and produce signaling activity, inverse agonists stabilize the receptor and inhibit basal activity, and antagonists block the receptor and produce no activity: Binding of an agonist or a partial agonist would lower the energy barrier from the inactive state to the active state and/or stabilize the active state, shifting the equilibrium toward the active state, while binding of an inverse agonist would enhance the energy barrier and/or stabilize the inactive state, shifting the equilibrium toward the inactive state. Not only ligand binding but also coupling to cognate G proteins would modify the equilibrium between the inactive state and the active state . In addition to ligands or G proteins that contribute to receptor stability, altering receptor residues involved in receptor activation (e.g., constitutively active mutations (CAMs) that increase the basal activity of the receptor in the absence of a ligand (for reviews, see [12-14]) is an alternative way to modify the equilibrium toward the active state.
It should be noted that structurally diverse ligand classes of a GPCR can induce the ligand-specific conformational changes in the receptor, determining different receptor states that are capable of activating specific subtypes of cognate G protein, as proposed in the protein ensemble theory describing proteins as a collection of conformational states . In fact, evidence from many biophysical studies supports the existence of multiple, ligand-specific conformational states of GPCRs [16-19]. In addition to ligand-specific receptor conformations, it has been indicated that the agonist bound GPCRs can have multiple, state-specific conformations corresponding to different signaling states [20,21]. For example, it is known that two distinct forms of the photoactivated rhodopsin, MII and MIIa/MIIb have been reported [22-24] and that the agonist-bound β2 adrenergic receptor (β2AR) exists in at least two different conformational states [25,26].
To date, the X-ray structures available include rhodopsin [27-37], β1AR , β2AR [39-41], and the adenosine A2A receptor (AA2AR) . In spite of the overall same topology, these X-ray structures show several interesting differences in local regions , as illustrated in the comparison of β2AR with rhodopsin: 1) H1 of rhodopsin is packed closely to the helical bundle because of a kink in the middle of the helix, while H1 of β2AR is loosely packed to the helical bundle without the equivalent kink; 2) The N-terminal of rhodopsin is ordered with β-sheets stacked on the top of E2 covering the ligand binding pocket, while that of β2AR is disordered; 3) The binding pocket in rhodopsin with a β-sheet alignment of E2 is totally blocked, while that in β2AR with an α-helical segment within E2 is only partially blocked for easy solvent or ligand access ; 4) The X-ray structure of rhodopsin  represents the inactive state, while that of β2AR-T4L fusion protein  represents an active-like state [44,45].
It appears that a receptor conformational change suitable for G protein activation does not require a dramatic change in the whole receptor but a change in the cytoplasmic side. For example, the X-ray structure of β2AR-T4L fusion protein , an active-like state, shows little difference from the X-ray structure of rhodopsin (inactive state) . Similarly, compared with the inactive form of rhodopsin , the photoactivated state metarhodopsin II (MII)  showed surprisingly little change, except an increased disorder in I3. This result suggests that photoactivation results in I3 flexibility. Evidence not only from the X-ray structures of GPCRs but also from many biophysical studies supports the conformational change in the intracellular side [47-51], including the breakage of the salt bridge between R3.50 and E(D)6.30 of the receptor, proposed as the ionic lock , that maintains the inactive state of the rhodopsin class GPCRs mainly through H6 stabilization but needs to be cleaved upon receptor activation. Elling et al.  proposed a global toggle switch activation model, where upon activation the receptor changes conformation such that the intracellular segments of H6/H7 move outward and the highly conserved TM Pro in the middle of H6 and H7 act as the pivot for the vertical seesaw movements. (In this review, a numbering system similar to Ballesteros-Weinstein system  is used for all the amino acids. For example, R1353.50 represents Arg135 with the highest conservation, indicated by the number 50, in the TM helix 3, indicated by the number 3. Similarly, V173E2 represents Val 173 of the second extracellular loop.)
All the X-ray structures are the antagonist or inverse agonist bound forms: for rhodopsin, retinal (inverse agonist) ; for β2AR, carazolol (partial inverse agonist) ; for β1AR, cyanopindolol (antagonist) ; and for AA2AR, ZM241385 (antagonist) . In considering the roles of the antagonist in blocking the receptor and the inverse agonist in stabilizing the inactive form of the receptor, it would be natural to have the X-ray structures of antagonist or inverse agonist bound GPCRs compared to those with agonist bound GPCRs, which is short-lived for G protein signaling. It has been argued that the X-ray structures of GPCRs are unsuitable for screening partial or full agonists [54-56]. Thus, if the screening of novel compounds is directed by using a receptor structure that is from its complex with an antagonist or inverse agonist, one may end up with antagonists or inverse agonists rather than with agonists. For example, Kolb et al.,  used the X-ray structure of β2AR to dock approximately 1 million commercially available small molecules and identified some compounds with relatively high affinities, most of which showed inverse agonist activity, suggesting that the receptor conformation of the reported X-ray structure of β2AR is adapted to mostly recognize the inverse agonist.
Thus, it seems apparent that the receptor structure of the agonist-bound form, at least, is necessary for drug design of agonists, though without knowing the protein structure of the high-affinity state GPCRs in complex with a G-protein [58,59] the picture is incomplete to fully understand the receptor in its active state to which an agonist preferentially binds. However, the agonist bound GPCR structures, are extremely difficult to obtain . Theoretically it is possible to obtain the receptor in its active state by starting from the inactive state of the receptor along the reaction path allowed for this conversion. However, the current state of the art MD simulations reported in the microsecond time scale [60,61] are not sufficient to provide the molecular details of the active state of a GPCR that would form in the millisecond scale . In addition, this path, largely unknown, contains multiple binding motifs for diverse ligands and multiple active conformers of the receptor. In this regard, recent studies [62-71] reported possible early events along this path from the inactive state toward the active state.
Brain CB1 cannabinoid receptors  are GPCRs and belong to the rhodopsin-like subfamily . The CB1 receptor is coupled to G-proteins for signal transduction pathways that inhibit adenylyl cyclase activity and regulate ion channels [72-76]. As shown in Fig. (1), the sequence alignment of the CB1 receptor with some GPCRs whose X-ray structures are available indicates that they share common features, including the seven TM helices and the highly conserved functional motifs, suggesting that the CB1 receptor has a similar molecular mechanism for receptor activation as for other rhodopsin class GPCRs. Based upon the phylogenetic study by Joost and Methner , where 241 human GPCRs were divided into 19 subgroups (A1 through A19), it was shown that the CB1 receptor belonged to subgroup A13 next to subgroup A17 from which AA2AR and βARs were branched and that these receptors altogether belonged to a larger cluster different from subgroup A16 to which rhodopsin belonged. Similarly, Fredriksson et al.  performed phylogenetic analyses of 342 unique functional non-olfactory human GPCRs to obtain five main families, including glutamate, rhodopsin (α, β, γ, and δ groups), adhesion, frizzled/taste2, and secretin. Further, the α-group of the rhodopsin family was classified into several distinct clusters, including the prostaglandin cluster, the amine receptor cluster, the opsin receptor cluster, the melatonin receptor cluster and the MECA (melanocortin, endothelial differentiation, cannabinoid and adenosine binding) receptor cluster to which cannabinoid receptors belong. Interestingly, both phylogenetic analyses [77,78] revealed that some of the closest receptors to the cannabinoid receptors are the melanocortin (MC) receptors. Both receptors, commonly lacking the conserved disulfide linkage between H3 and E2, common to most rhodopsin class GPCRs [79,80] and important for receptor structure and activity , exhibit a high degree of basal activity [82,83], suggesting a role of the flexibility of E2 in basal activity. Interestingly, the MC-4 receptor (MC4R), which exhibits the highest sequence homology to the CB receptors (approximately 80 %) among the MC receptors, is known to be associated with 6 % of early onset obesity , indicating their common roles in regulating obesity. In addition to the traditional cannabinoid receptors by which agonist activation is mediated through Gi/o protein , a novel putative cannabinoid receptor GPR55 has recently been characterized [86,87]. According to the GPCR classification by Fredriksson et al. , GPR55 belonged to the δ-group of the rhodopsin family within the purine receptor cluster, which is different from the α-group to which the cannabinoid receptors belong.
Mutagenesis data for the cannabinoid receptors have been accumulated for more than a decade. The mutagenesis analysis, often combined with the computational modeling analysis, provides invaluable insights into characterizing the role of the CB1 receptor residues in ligand binding and receptor activity. However, it is not always straightforward to interpret the effects of the mutated residue due to the conformational complexity inherent to GPCRs  as described earlier. This is especially true for the case of the cannabinoid receptors that exhibit a high level of basal activity [82,88-90]. Thus, Picone et al.  writes, “Introduction of a point mutation within a receptor binding site has the potential of altering ligand binding properties. However, it could also confer undesirable and frequently unpredictable consequences on the global conformation of the receptor that may affect ligand binding in a manner unrelated to binding site structure.” In the same context, Beukers and Ijzerman  describe, “By the technique of site-directed mutagenesis by which point mutations are introduced, the role of specific residues in receptor structure and function can be easily studied. However, careful analysis of mutagenesis data is required because the replacement of an amino acid can result in a local effect as a result of a gain or loss of interaction with neighboring residues or in a global effect such as an alteration of protein folding or protein stability”.
In this review, I divide the CB1 receptor functional residues into three different types, ligand binding (B), receptor stabilization (S) and receptor activation (A) residues, according to their roles as suggested from the mutational studies. Effects of the mutations of these residues are schematically shown in Fig. (2). It is interesting to note that all these residues can contribute to receptor activation. It should be noted that it is often difficult to define the function of a residue as one specific type over the others due to the fact that their roles are often closely associated with each other.
The type Bd residues form the binding pocket and directly contact with the ligand (Fig. (2)). Thus, mutations of the type Bd residues alter binding site structure and affect ligand binding by disrupting specific ligand interactions. This type of residues are useful not only for describing the binding pockets distinct to the individual classes of ligands but also for delineating the early stages of receptor activation (see below).
The type S residues stabilize the receptor structure  (Fig. (2)). Mutations of this type of residues may cause the failure of receptor expression due to misfolding and abolish the ligand binding affinity and receptor activation. An example of the type S residue is illustrated by W4.64 (i.e., the Trp residue at the 4.64 position ) of the cannabinoid receptors that is known to be critical for ligand binding and signaling . It was reported that the mutation of W2554.64 of the CB1 receptor resulted in the failure of receptor expression [95,96], suggesting that W4.64 is important for receptor structure. Combining these results with the finding that the highly conserved W4.64 in many GPCRs is known to be important for receptor folding , W2554.64 of the CB1 receptor is a type S residue important for receptor structure.
If a type S residue affects ligand binding by indirectly modifying the binding pocket topology, it can be further classified into BiS residues important for the binding of inverse agonists that stabilize the receptor. Thus, mutations of the type BiS residues result in alteration in ligand binding and impair ligand binding and receptor activation (Fig. (2)). In fact, many residues reported to be important for ligand binding belong to this type of residues (see below).
The type A residues play an important role in receptor activation by being directly involved in receptor activation (Fig. (2)). Thus, mutations of the type A residues directly alter G protein signaling. Examples of the type A residues are seen in CAMs which decrease the inverse agonist binding and increase basal activity [13,98]. Another example is seen from those residues associated with the D(E)RY motif known to be crucial for G-protein activation , by the formation of the ionic lock , without participating in ligand binding. Thus, Song and his colleagues  reported that D6.30N mutation of the CB receptors maintained the ligand binding affinity but exhibited the greatly reduced signaling activity, suggesting that these residues are the type A residues.
If the type A residues indirectly affect ligand binding through modifying the binding pocket topology, they can be further classified into BiA (Fig. (2)). An example of the type BiA residue was illustrated by Song and Feng  in their site-directed mutagenesis study showing that Y2095.58A mutation of the peripheral cannabinoid (CB2) receptor  reduced the binding affinity of 2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl] (1-naphthyl)methanone (WIN55212-2), (6aR,10aR)-9-(Hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol (HU210) and N-arachidonoylethanolamine (anandamide) by 5- to 8-fold but abolished signaling activity. Although this study was done on the CB2 receptor, considering that Y5.58 is important for breaking the ionic lock  and located deep down the core and not involved directly in ligand binding, it is likely that the conserved Y2945.58 of the CB1 receptor is a type BiA residue for cannabinoid agonists.
It was shown that the L3416.33A/A3426.34L double mutation of the CB1 receptor exhibited decreased (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclo-hexan-1-ol (CP55940) binding by 4-fold an partial constitutive activation (not Gi but Gs)  and that D1303.49A, R1313.50A and A2446.34E mutations of the CB2 receptor abolished agonist-induced G-protein activation with retaining the binding affinity of anandamide, HU210 and WIN55212-2 only by R1313.50A mutation . Considering all these residues are conserved in both the CB1 and CB2 receptors (Fig. (1)), it is likely that R1313.50 is a type A residue without interfering in ligand binding, while D2133.49 and A3426.34 of the CB1 receptor are BiA residues for anandamide, CP55940 and WIN55212-2.
It was shown that T2103.46I and L2073.43A mutations of the CB1 receptor increased the binding affinity of methanandamide, a synthetic analog of anandamide known to be metabolically stable, by 6-fold and 3-fold, respectively [105,106]. It was shown that these mutations increased CP55940 binding affinity by 3-fold, decreased WIN55212-2 binding by 3-fold and decreased N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) binding by about 30-fold, suggesting that these residues are CAMs. With the location of these residues near the DRY motif far away from the binding site, these residues are less likely to directly interact with the ligand but play an important role in regulating the receptor conformation in association with G protein activation. Thus, it is likely that they are BiA residues for anandamide, CP55940, WIN55212-2 and SR141716A.
It was shown from a recent study combining computational and mutational analyses that anandamide exhibited an approximately 13-fold decrease in binding affinity by the Y2755.39F mutation of the CB1 receptor and that the Y2755.39I mutation abolished ligand binding and receptor signaling . These results suggest that Y2755.39 is important for anandamide binding, possibly through H-bonding. The authors concluded that the Y2755.39I mutation altered ligand interactions within the binding site. Thus, it is likely that Y2755.39 of the CB1 receptor is a Bd residue for anandamide. However, it is possible that the Y2755.39I mutation may cause a global effect  resulting in a significant modification in the ligand binding pocket. In support, it was reported that Y2755.39 of the CB1 receptor and Y1915.39 of the CB2 receptor were not direct ligand contact sites [108,109] and that the Y2755.39S and Y2755.39A mutations resulted in failure of receptor expression .
No mutations of F2003.36A, W2795.43A and W3566.48A, but F1893.25A moderately (approximately 6-fold) decreased anandamide binding affinity . Illustrated by an anandamide docking model developed from these obtained mutation data, the C5-C6 double bond of the ligand interacted with F1893.25 via aromatic/π interaction . These results indicate that F1893.25 is a Bd residue for anandamide.
McAllister et al.  showed that CP55940 binding to the CB1 receptor was retained by the Y5.39F mutation but abolished by the Y5.39I mutation, suggesting that the aromaticity of the residue at the 5.39 position is crucial for CP55940 binding. The authors concluded that altered interactions caused by the Y5 39I mutation resulted in alteration in ligand binding. Thus, it is likely that Y2755.39 of the CB1 receptor is a Bd residue for CP55940.
It was shown that the Ala mutations of the E1 residues (H181, R182, K183 and D184) and the H3 proximal residues (V1883.24 and F1893.25) resulted in reduced binding affinities of CP55940, but not of SR141716A . With decreases in CP55940 binding less than expected by direct ligand contact residues, the results suggest that the effects are not due to direct contact with the ligand but due to loop structural alteration that indirectly affects ligand binding through modifying the binding pocket topology. Thus, these residues on the extacellular side of the receptor are BiS residues for CP55940. Interestingly, for the binding affinity of CP55940 by the F1893.25A mutation of the CB1 receptor, Murphy and Kendall  reported an approximately 60-fold decrease, but McAllister et al.  reported a 3-fold decrease.
It has been shown from a combined study of affinity labeling, site-directed mutagenesis, and ligand docking studies  that a classical cannabinoid derivative having the reactive electrophilic isothiocyanate moiety at the end of the C3 side chain formed a covalent bond to the nucleophilic C3556.47 and retained the binding affinities to the C3556.47A and C3556.47S mutants but had a reduced affinity to the C3556.47L mutant. No change in WIN55212-2 binding affinity for any of mutant receptors was shown. These results suggest that C3556.47 of the CB1 receptor is part of the binding site of the classical and non-classical cannabinoids, distinct from that of WIN55212-2, and is involved in receptor activation as part of the highly conserved functional CWxP motif [112,113]. Thus, C3556.47 of the CB1 receptor is a Bd residue for CP55940.
In contrast to the profound effect the S3837.39A mutation of the CB1 receptor produced upon CP55940 binding, the binding of WIN55212-2 and SR141716A was unchanged by this mutation . Rhee observed that the S2857.39A mutation of the CB2 receptor resulted in dropping HU243 binding approximately 13-fold , suggesting that the conserved S7.39 of the CB receptors are involved in CP55940 binding. Demonstrating by computational modeling studies that the S3837.39A mutation reduced the H7 kink, Kapur et al.  proposed that S3837.39 induces a bend in the extracellular side of H7 required for CP55940 binding. It is interesting to note that the mutation of the equivalent residue A2927.39 of rhodopsin resulted in a constitutively active receptor . Taken together, it is likely that S3837.39 is a BiA residue for CP55940.
In a very recent Ala scanning mutagenesis study, Kendall and her colleagues  comprehensively analyzed the E2 of the CB1 receptor and demonstrated that the Ala mutations of both the N-terminal and C-terminal residues abolished CP55940 binding and G protein signaling. Interestingly, Ala mutations of P269E2, H270E2 and I271E2 were insensitive for SR141716A binding. As shown from the accompanied homology molecular model, these hydrophobic residues were deeply inserted into the core and formed part of the ligand binding site, suggesting that they are CP55940-specific residues. Together, W255E2 and N256E2 are S residues, while F268E2, P269E2, H270E2 and I271E2 are Bd residues for CP55940.
From a mutagenesis study , it was shown that the CB1 D1632.50N mutation reduced WIN55212-2 binding by approximately 400-fold, but not for other cannabinoid ligands. It was also shown that the mutated receptor exhibited greatly attenuated inhibition of cyclic AMP production by both CP55940 and WIN55212-2. These results strongly suggest that D1632.50, a highly conserved residue in many GPCRs, is important in the CB1 receptor for WIN55212-2 binding and receptor activation. As the authors discussed, the D1632.50N mutation may cause the receptor to alter the binding pocket uniquely defined for WIN55212-2. Thus, it is likely that D1632.50, without directly interacting with the ligand, is a BiA residue for WIN55212-2. However, it should be noted that Mackie and his colleagues, in contrast, reported that D1642.50 in the rat CB1 receptor had no effect on ligand binding or inhibition of cyclic AMP production but did alter signaling via Gi by WIN55212-2 .
It was shown from the CB1 chimeric receptor formed by the replacement of H3 with that of the CB2 receptor  that H3 residues were important for WIN55212-2 binding and that the G1953.31S mutated CB1 receptor exhibited an enhanced WIN55212-2 binding by about 5-fold. The authors proposed that the enhanced WIN55212-2 binding affinity was attributed to H-bonding between the carboxyl oxygen of the ligand and S3.31 of the mutated receptor, based upon the report that AAI analogs lacking the carboxyl oxygen displayed a 7- to 10-fold decrease in binding affinity . Thus, it is likely that G1953.31 of the CB1 receptor is a Bd residue for WIN55212-2.
It was shown from a study combining molecular modeling and site-directed mutagenesis approaches  that the V2825.46F mutation of the CB1 receptor enhanced the WIN55212-2 binding affinity by 12-fold; this mutation did not change the binding affinity of cannabinoid compounds and anandamide, suggesting that V2825.46 is a Bd residue for WIN55212-2 binding .
McAllister et al.  showed that WIN55212-2 binding was retained by the Y2755.39F mutation of the CB1 receptor but abolished by the Y2755.39 I mutation, suggesting that the aromaticity of the residue at the 5.39 position is crucial for WIN55212-2 binding. Thus, it is likely that Y2755.39 of the CB1 receptor is a Bd residue for WIN55212-2.
It was shown that the F2003.36A and W2795.43A mutations of the CB1 receptor reduced the binding of WIN55212-2 by 9-fold and 16-fold, respectively, but did not affect CP55940 binding , leading to proposing the importance of an aromatic cluster within the CB1 receptor aromatic microdomain formed by H3-H4-H5-H6 for WIN55212-2. Similarly, the F2003.36A mutation of CB1 showed a moderate decrease in WIN55212-2 binding but no change in CP55940 binding . Thus, these residues are Bd residues for WIN55212-2.
It was shown that the W2795.43A or W3566.48A mutation of the CB1 receptor reduced significantly the binding of SR141716A , suggesting the important of these residues for the binding of SR141716A. Thus, these residues are type Bd residues for SR141716A.
It was shown that the mutation of E2 Cys residues C257E2 and C264E2 to Ala abolished the binding affinity of SR141716A , implying a significant conformational change, possibly due to breaking the intra-loop disulfide bond and a modification of the ligand binding pocket. In the same study, it was also shown that the introduction of a bulky group on C3867.42 inhibited SR141716A binding , suggesting that C3867.42 directly interacts with SR141716A. Thus, C257 and C264 are BiS residues, while C3867.42 is a Bd residue for SR141716A.
From site-directed mutagenesis studies of the human CB1 receptor [122,123], it was shown that the K1923.28A mutation resulted in a complete loss of binding and a significant reduction in receptor activity for HU210, CP55940 and anandamide, but retained binding and receptor activity for WIN55212-2. It was shown that a similar binding affinity decrease occurred (17-fold) for SR141716A in the K1923.28A mutant as was seen (14-fold) for a SR141716 analog lacking the C3 carboxamide oxygen binding in the wild type receptor [124,125]. Thus, the current understanding of ligand binding to the CB1 receptor is heavily dependent upon the generally accepted role of K1923.28 as one of the key residues that directly interact with CP55940 [122,126] and SR141716A [124,125] but not with WIN55212-2. Thus, most of the computational docking studies of the CB1 receptor [91,95,114,128-133] employed K1923.28 as the primary interaction site for the phenolic OH of the cannabinoid ligands through H-bonding.
However, as opposed to the generally accepted hypothesis that K1923.28 residue directly contacts with the ligand (i.e., as a Bd residue), there have been a number of findings indicative of a role of K1923.28 in indirectly modifying the binding pocket geometry (i.e., as a BiS residue). First, the effect of the K1923.28A mutation of the CB1 receptor on ligand binding was, in fact, universal to all the ligands: anandamide and CP55244 were very sensitive and exhibited no binding, SR141716A binding was moderately sensitive to exhibit a 17-fold decrease , and WIN55212-2 was less sensitive and exhibited about 2-fold decrease [122,123]. This can be interpreted as follows: K1923.28A mutation leads to an indirect modification of the ligand binding pocket geometry such that the binding of anandamide and CP55244 is more severely disrupted than the binding of WIN55212-2. Second, it was observed from the mutation study by Chin et al.  that CP55940 showed no difference in binding affinity for the wild type and the K1923.28R CB1 receptor. This finding suggests that K1923.28 is not a Bd residue for CP55940, for if K1923.28 directly interacts with CP55940 the binding should be less favored by the bulkier replacement. Third, the removal of the phenolic OH of CP55940 showed only a 35-fold drop in ligand binding but a 200-fold decrease in receptor activity . This drop in ligand binding is much lower than expected  for deletion of the direct H-bond between the charged N of K1923.28 and the phenolic OH of the ligand. Fourth, according to a recent CB1 receptor homology model embedded in a lipid bilayer , it was shown that K1923.28, as the charged form at physiological conditions, was snorkeling [137-139] by stretching out its long side chain N atom for the formation of a strong salt bridge with D184E1 near the polar membrane surface, while continuing to surround the side chain hydrocarbon moiety with several hydrophobic residues from H2 and H3 inside the lipophilic membrane. It is conceivable, however, that K1923.28 can directly interact with the ligand if the ligand contains a suitable functional group, such as a carboxylate or phenoxy moiety, or a π-electron rich moiety (i.e., an aromatic ring) that satisfies the positively charged N side chain of K1923.28 in the hydrophobic lipid environment by forming a H-bond or π-cation interaction [140,141] by the replacement of the existing salt bridge with D184E1.
Taken altogether, it is possible that K1923.28 can play roles in ligand binding not by directly contacting with the ligand but by indirectly modifying the binding pocket geometry. With its role remaining to be further confirmed for a better understanding of the CB1 receptor-ligand interaction, which is crucial not only for studying receptor activation but also for developing CB1 receptor structure-based drugs, K1923.28 is considered in this review as a BiS residue for the CB1 receptor ligands.
The functional residues of the CB1 receptor involved in ligand binding (Bd,BiS and BiA) are listed in Table 1. Based upon the CB1 receptor residues defined as the types Bd that are directly involved in contacting with the ligand, the binding pockets of anandamide, CP55940, WIN55212-2 and SR141716A can be approximately delineated. Among these residues identified as key binding site residues, it is interesting to note that F1893.25  is uniquely defined for anandamide, F268E2-I271E2  and C3556.47  for CP55940, G1953.31  and V2825.46  for WIN55212-2, and W3566.48 and C3867.42  for SR141716A (Table 1 and Fig. (3)). These results indicate that, with some overlap in common structural features for CB1 receptor binding, each class of structurally diverse ligands exhibits unique interactions with the receptor  for ligand-specific effects on signaling activity. Considering the fact that an agonist preferentially binds to the high-affinity state receptor (i.e., an active state receptor), where the rigid-body movements occur mainly in H6 and H7 according to the global toggle switch model , the Bd residues on H6 and H7 of the inactive receptor are not included as initial contacts for agonist binding but become available when the receptor is fully activated by the inward movements of H6 and H7 [50,103]. Thus, it is likely that C3556.47  is not an initial contact residue of the receptor in the inactive state with CP55940 (Fig. (3B)). In contrast, the Bd residues on H6 and H7 for SR141716A (e.g., W3566.48  and C3867.42 ), positioned toward the binding core without rigid-body movements of H6 and H7, are directly involved in the ligand binding (Fig. (3D)) and stabilize the inactive receptor. In this regard, the competitive ligand binding between the CB1 receptor agonists and SR141716A should be viewed in terms of not only competing with the ligand binding pocket but also competing with shifting the equilibrium between the inactive state and the active state.
Only a few residues, including F1893.25  and Y2755.39 [107,110], are known to directly interact with anandamide. Taken the H2/H3/H6/H7 region proposed to be important for anandamide binding  together with the exclusion of H6 and H7 due to their involvement in agonist binding at the late stage of receptor activation, the key initial contacts for anandamide binding would be in the H2-H3-H5 region (Fig. (3A)).
For CP55940, F268E2/P269E2/H270E2/I271E2 , Y2755.39  and C3556.47  are directly contacting with the ligand. A very recent substituted-cysteine accessibility method (SCAM)  study on H6 of the CB2 receptor  showed that V2616.51, L2626.52, L2646.54, M2656.55, L2696.59 and T2726.62 were on the solvent-accessible surface of the binding site crevice of CP55940, suggesting that the equivalent residues in the CB1 receptor are accessible to CP55940. Taken the region formed by H3-H5-H6-H7 proposed to be important for CP55940 binding [114,144] together with the exclusion of H6-H7 due to their involvement in agonist binding at the late stage of receptor activation, the key initial contacts for CP55940 binding would be in the H3-E2-H5 region (Fig. (3B)).
For WIN55212-2, G1953.31 , F2003.36 [95,120], Y2755.39 , W2795.43  and V2825.46  are the residues in direct contact. Taken the proposed H3-H4-H5-H6 region for WIN55212-2 binding [95,145] together with the exclusion of H6/H7 due to their involvement in agonist binding at the late stage of receptor activation, the key initial contacts for WIN55212-2 binding would be in the H3-H4-E2-H5 region (Fig. (3C)).
For SR141716A, F2003.36 , W2795.43 , W3566.48  and C3867.42  are the residues in direct contact. It has been suggested that E1 and H3 (proximal to the extracellular side)  and E2 [96,110] are not sensitive to SR141716A binding. Taken the proposed H3-H4-H5-H6 region for SR141716A binding  together with the inclusion of H6/H7, the key contacts for SR141716A binding would be in the H3-H5-H6-H7 region (Fig. (3D)). It is interesting to note that the binding site of SR141716A is located at W3566.48 , in the region deep in the receptor core and relatively distal to the extracellular side (see Fig. (3)), possibly protecting the receptor from activation.
The second extracellular loop (E2) connecting H4 and H5 appears to play an important role in receptor stabilization, ligand binding and receptor activation [16,17,79,146-148]. It is shown from the GPCR X-ray structures that E2 residues form part of the binding site for the ligand. It has been suggested that different E2 residues are involved in different functions [149,150], possibly with distinct conformations [16,19]. It has been suggested from several studies [96,110,121,145,151] that E2 of the cannabinoid receptors are important for ligand binding and receptor activity. It is known that E2 Cys residues C257E2 and C264E2 of the CB1 receptor are required for receptor stabilization [110,121]. It was shown from an early study by Martin and his colleagues  that CP55940 binding was inhibited competitively by sulfhydryl blocking agents but not by a disulfide reducing agent, suggesting that at least one reactive sulfhydryl group exists at the CP55940 binding site and that a disulfide bond, whose reduction impacts on CP55940 binding, exists away from the CP55940 binding site. Among the Cys residues conserved in the cannabinoid receptors, C3556.47 would be the best candidate to provide the reactive sulfhydryl group with the following reasons: First, C3556.47 is known to form part of the CP55940 binding site ; Second, it was shown from a mutational analysis  that the CB1 receptor mutations of C257E2 and C264E2 did not affect CP55940 binding; Third, it was shown that sulfhydryl blocking of C3867.42 did not affect CP55940 binding . Accordingly, it can be argued that although the CB1 receptor lacks the inter-disulfide linkage between H3 and E2, two E2 Cys residues form an intra-loop disulfide bond [110,121,145] important for CP55940 binding  as well as receptor stabilization [110,121].
It has been shown that the C-terminal of E2 of many GPCRs is important for ligand binding and receptor function [146,150,153]. It is also shown from the GPCR X-ray structures of rhodopsin  and β2AR [40,154] that an aromatic residue of the Cys-X-X-X-Ar motif, where Cys is tied to another Cys from H3, conserved in many GPCRs , plays a role in receptor stabilization and ligand binding. The importance of the C-terminal region of E2 shown by many GPCRs has also been demonstrated in the CB1 receptor . Thus, it was shown from a recent Ala scanning mutagenesis of E2 of the CB1 receptor  that the C-terminal residues F268E2-I271E2 were critical for CP55940 but not for SR141716A binding, suggesting that the C-terminal end of E2 plays an important role not only in ligand binding but also in receptor activation.
It has been suggested that E2 plays an important role in receptor activation, primarily by coupling to the TM helical domain [8,155,156]. It appears that E2 rearrangement triggered by the ligand is coupled to the TM helical domain, especially the segments of H5  and H7  to achieve receptor activation. For the CB1 receptor, it was suggested from a recent study  that the C-terminal end of E2 of the CB1 receptor played an important role in receptor activation, presumably through the coupling to H5 similar to rhodopsin . It is possible that the Bd residues in the extracellular side of the H5 through H7 region [95,144], including Y2755.39 , W2795.43 , V2825.46  and C3556.47  of the CB1 receptor (Table 1), would be involved in providing the necessary movement in the TM helical region for receptor activation.
Activation of a GPCR is a multistep process [11,157] that is initiated by binding of an agonist to the inactive state of the receptor. Ahuja and Smith  proposed a mechanism of GPCR activation by multiple sequential switches, including a switch of E2 displacement, a switch of the H5 motion coupled to E2, a rotamer toggle switch of the CWxP motif on H6, and an internal switch of the NPxxY motif on H7, the final switch that eventually breaks the ionic lock between R3.50 and E6.30 on the intracellular sides of H3 and H6 to relay the molecular signal to the coupled G protein. Thus, according to this mechanism, the molecular signal from the extracellular side passes through the receptor in the following order: E2 -> H5 -> H6 (the rotamer toggle switch) -> H7 (the internal switch) -> H3/H6 (the ionic lock). As shown in Fig. (4A), most of these residues involved in receptor activation (i.e., A and BiA residues) identified from the mutational studies of the CB1 receptor are, in fact, part of the functional motifs conserved in many GPCRs  (see Fig. (1)). R2143.50 , D3386.30 , L3416.33/A3426.34  and T2103.46/L2073.43 [105,106] are associated with the DRY motif; and D1632.50 [116,160] of the SLAxAD motif. Although C3556.47 [112,113] and W3566.48  of the CWxP motif are classified as Bd residues, they also play an important role in receptor activation. It was shown in a recent CB1 receptor helical bundle model  that most of the functional motifs were involved in maintaining the inactive state of the CB1 receptor, suggesting that disruption of these functional motifs is necessary in order to achieve a conformational change of the receptor necessary for G-protein signaling [49,161].
It has been proposed that upon receptor activation an outward movement of the extracellular side of H5, coupled to an outward displacement of E2 , releases the molecular constraints on W6.48 , while an inward movement of the intracellular H5 replaces E6.30 by Y5.58, disrupting the ionic lock . It has also been proposed that an outward movement of the intracellular side of H6, as a result of the disruption of the rotamer toggle switch , is one of the key steps in receptor activation . The X-ray structure of rhodopsin  reveals that W2656.48 is stabilized by aromatic stacking with F2616.44 and Y2686.51, highly homologous residues in rhodopsin class GPCRs , and by the water-mediated H-bond interaction with N3027.49 of the NPxxY motif . It has been suggested that the breakage of the H-bond between W2656.48 and N3027.49 allows M2576.40 to move away and Y3067.53 to come in contact with H6, contributing to breaking the ionic lock . Thus, it appears to be crucial to see how W3566.48 is stabilized in the CB1 receptor inactive state for understating the mechanism of the CB1 receptor activation. The CB1 receptor lacks aromatic residues at the 6.44 and 6.51 positions, and consequently is expected to have unique interaction patterns for W3566.48. As shown in Fig. (4B), it was revealed from a recent homology model of the CB1 receptor in the inactive state  that W3566.48 formed aromatic stacking with F2003.36 and F1702.57 and was involved in an extensive H-bond network by direct H-bonds with C3867.42/N3897.45. It was also shown that W3566.48 was conserved by a water-mediated H-bond network by C3556.47/L3596.51/C3827.38/C3867.42, located just above W3566.48 (Fig. (4B)). The importance of the aromatic stacking between W3566.48 and F2003.36 is supported by the proposed rotamer toggle switch by W3566.48/F2003.36 of the CB1 receptor [165; 112], similar to W2866.48/F2906.52 of β2AR  and also by the finding that the F200A mutation of the CB1 receptor resulted in higher constitutive activity compared with the wild type receptor . Thus, alternative ways to release the molecular constraints on W3566.48 of the CB1 receptor to achieve CB1 receptor activation include: 1) the breakage of aromatic stacking between W3566.48 and F2003.36/F1702.57; and 2) the breakage of a water-mediated H-bond network by C3556.47/L3596.51/C3827.38/C3867.42.
It was also shown in a recent CB1 receptor model  that N3937.49 of the NPxxY motif formed direct H-bonds to N3897.45 and D1632.50 (Fig. (4B)). Thus, similar to rhodopsin, the breakage of the N3897.45-mediated H-bond between W3566.48 and N3937.49 contributes to breaking the ionic lock in the CB1 receptor. It has been proposed that L2073.43, one of the highly conserved residues among many GPCRs , maintains the receptor in the inactive state by locking the movement of H6. In support of this, L2073.43 of the CB1 receptor has been reported to be a CAM residue . It is possible that an outward movement of the intracellular side of H6 allows I3486.40, similar to M2576.40 in rhodopsin , to move away from L2073.43, thereby causing N3937.49 to come in contact with H6 (Fig. (5)). Taking into consideration the role of D1632.50 in receptor activation , it is possible that the breakage of the interaction between N3937.49 and D1632.50, which is proposed as an activation switch in other GPCRs [166-168], would contribute to breaking the ionic lock. It also appears that upon receptor activation Y2945.58 and Y3977.52 of the CB1 receptor, both highly conserved in many GPCRs, come closer and contribute to the breakage of the ionic lock (Fig. (5)).
Supported by similar results from other GPCR systems , several studies on the CB1 receptor [170,171] suggest that different CB1 receptor ligand classes evoke selective G-protein signaling. Considering the concept that one receptor can couple to different signaling pathways by different ligands , the binding of structurally distinct CP55940 and WIN55212-2 contributes to the ligand-specific conformational change in the receptor . In this regard, it is insightful to see that among the residues of the CB1 receptor involved in the ligand-induced receptor activation (i.e., Bd residues), F268E2-I271E2  and C3556.47  are unique to CP55940, while G1953.31 , F2003.36 [95,120], W2795.43  and V2825.46  are unique to WIN55212-2 (Table 1). Thus, these unique residues play important roles in inducing ligand-specific receptor activation. Considering the suggested important role of aromatic stacking in receptor activation [95,113,157,172], it is likely that F268E2  and F2003.36 [95,120] are important for receptor activation by CP55940 and WIN55212-2, respectively. Thus, employing the known type Bd residues, albeit limited in number, it is possible to describe the ligand-specific interference with the CWxP motif as the key step of the CB1 receptor activation mechanism [112,165]. Here, the ligand-specific CB1 receptor activation is illustrated by comparing CP55940 with WIN55212-2.
For CP55940, the Bd residues include F268E2/P269E2/H270E2/I271E2 , Y2755.39  and C3556.47 . It has been suggested that the E2 C-terminal F268E2 is crucial for CP55940 binding but not for that of SR141716A . It has also been suggested that Y2755.39 is crucial for receptor activation by CP55940 . Thus, upon CP55940 binding, the aromatic stacking interaction between the ligand aromatic A-ring and the receptor residues, including F268E2  and Y2755.39 , composes the key initial contacts, which allows the hydrophobic C3 side chain of CP55940, the steric trigger for receptor activation , to position deep in the core. It is likely that the interaction between the C3 side chain of the ligand and L3596.51 on the solvent-accessible surface of the binding site crevice of CP55940  contributes to the breakage of the water-mediated H-bonds (Fig. (4B)), which, in turn, interferes with the extracellular side of H7 and with the CWxP motif (Fig. (5)). As a result, the molecular constraints on W3566.48 are released and W3566.48 becomes free to rotate for receptor activation. It is possible that the coupling movements of H5/H7 [8,155,156] by the ligand binding to E2  initially and then to the extracellular side of the H5 through H7 region [95,144] assist of the rigid-body rotation of H6 [52,174-179]. As shown in Fig. (3B), C3556.47 , whose side chain is pointing toward H7 and located off the receptor core region, is less likely to be accessible to CP55940 in the receptor inactive state, but becomes available for ligand binding with the rigid-body rotation of H6 upon receptor activation. The findings that C3556.47  and S3837.39  of the CB1 receptor were important for CP55940 but not for WIN55212-2 suggest that these residues play a role in CP55940-specific receptor activation. Overall, as shown in Fig. (5), the molecular signal by CP55940 binding from the extracellular side passes through the receptor in the following order: E2 -> H5/H7 -> H6 (the rotamer toggle switch) -> H7 (the internal switch) -> H6 (the ionic lock).
For WIN55212-2, the Bd residues include G1953.31 , F2003.36 [95,120], Y2755.39 , W2795.43 [95,120] and V2825.46 . Upon binding, WIN55212-2 interacts directly with Y2755.39  and W2795.43 [95,120] within the proposed aromatic microdomain  which allows the aromatic naphthyl ring, the steric trigger of WIN55212-2 for receptor activation (Shim and Howlett, 2006), to bind to F2003.36 of the proposed rotamer toggle switch of the CB1 receptor [112,165]. It is likely that the interaction between the naphthyl ring of the ligand and F2003.36 [95,120] contributes to the disruption of aromatic stacking between W3566.48 and F2003.36, interfering with the extracellular side of H3 and with the CWxP motif. As a result of the rotameric change in F2003.36, the molecular constraints on W3566.48 are released and W3566.48 becomes free to rotate for receptor activation. Because the water-mediated H-bonds above W3566.48 are in close proximity to F2003.36 (Fig. (4B)), it is possible that the water-mediated H-bonds are broken by WIN55212-2 binding to F2003.36. The findings that W2795.43 [95,120] and V2825.46  of the CB1 receptor were important for WIN55212-2 but not for CP55940 suggest that these residues play a role in WIN55212-2-specific receptor activation. Overall, the molecular signal by WIN55212-2 binding passes through the receptor in the following order: H5 -> H5/H3 -> H6 (the rotamer toggle switch) -> H7 (the internal switch) -> H6 (the ionic lock).
Interestingly, in order to disrupt the CWxP motif, CP55940 interferes with H7 and WIN55212-2 interferes with H3, through unique interactions with H5. Both paths of interference appear to be effective in disrupting the CWxP motif according to the global toggle switch model  where upon activation, W3566.48 of the CWxP motif was displaced with the outward movement of the intracellular segment of H6. The observed ligand-specific G-protein signaling is likely to be attributed different ways to disrupt W3566.48, through H5/H7 in CP55940 or through H5/H3 in WIN55212-2. It has been proposed that H5 plays an important role in agonist-specific conformational change . Thus, it is tempting to speculate that different paths of disrupting the CWxP motif contribute to the ligand-specific receptor activation.
Even before the X-ray structures of GPCRs became available, in their early pioneering works, Reggio and her colleagues  successfully determined the length and orientation of the membrane spanning 7 TM helices and the presence of H8 in the CB1 receptor. After the X-ray structure of rhodopsin  became available, this X-ray structure was utilized as the template to construct cannabinoid receptor homology models [95,124,128-130,132,182-184]. Recent CB1 receptor homology models [96,136] were constructed using the X-ray structure of β2AR  as templates. In spite of the similarity in overall structural topology, homology receptor models using these X-ray structures as templates may result in structures which are locally quite different. Although the CB1 receptor shows a low sequence homology to β2AR and rhodopsin (43 % and 42 % identical to human β2AR and bovine rhodopsin), homology models of the CB1 receptor using these GPCRs as templates have been justified by reliable sequence alignment with these GPCRs according to the highly conserved amino acid residues and functional motifs within the TM region (Fig. (1)), which are conserved in greater than 90 % of all GPCRs . Yuzlenko and Kieć-Kononowicz  tested the feasibility of the homology models of adenosine receptors using the X-ray structures of rhodopsin and β2AR as templates and concluded that the β2AR-based homology models were better than the rhodopsin-based homology models judging from the stability of the ligands inside the binding pockets. The feasibility of the homology models of the CB1 receptor, using either rhodopsin or β2AR as the template, remains to be seen. The CB1 receptor is 44 %, 45 %, 43 %, 42 %, and 45 % identical to AA2R (human), β1AR (turkey), β2AR (human), rhodopsin (bovine), and rhodopsin (squid), respectively. This suggests that the X-ray structures of AA2R , β1AR  and squid rhodopsin  would be better templates for constructing homology models of the CB1 receptor.
Some of recent homology models of the CB1 receptor [136,184,187] have taken advantage of increasing computational resources and were determined in a fully hydrated lipid bilayer to mimic the physiological environment. Such homology models of the CB1 receptor provide a detailed understanding of its interaction with the lipid bilayer, including hydrophobic core and hydrophilic interfaces, and with water . Some studies [95,132,184] also included CB1 receptor activated state models by modifying their receptor homology models in the inactive state on the basis of the receptor activation information obtained from biophysical studies [52,174-179]. On one hand, the active state of a GPCR is the state that perfectly fits to an agonist; a homology model of the CB1 receptor in its active state would better serve to understand the molecular interactions with an agonist. On the other hand, it is still very challenging to construct a computational model of a fully activated GPCR due to the reasons described earlier even with the large amount of accumulated biophysical data on the GPCR active state.
The X-ray structures of GPCRs and high quality homology models have been used for identifying potential lead compounds by the popular GPCR drug-design approach of structure-based high throughput screening (HTS) of a large collection of commercially available compounds [189-196] (for recent reviews, see [54,197]). Similarly, HTS has been applied to the cannabinoid receptor area [129,198,199]. A new drug-design approach, fragment-based approach (FBA) [200-202] has been successfully applied to screen relatively few compounds (approximately 1,000 compounds) using NMR or X-ray crystallography and to identify lead-like fragments with relatively low binding affinity (in the μM range) positioned non-redundantly at the binding pocket that can be connected to derive novel compounds with relatively high binding affinity (in the nM range). Compared with HTS, which needs an exhaustive search of the chemistry space allowed for the library of larger molecules, the chemistry space is efficiently probed by screening collections of small ligand fragments . FBA using NMR or X-ray crystallography cannot be applied widely to GPCRs owing to the difficulty in obtaining their X-ray structures, particularly for small fragments within the active site . To overcome this problem, a similar approach to FBA, multiple copy simultaneous search (MCSS)  can be applied to homology modeled GPCRs. MCSS generates several thousand replicas of a given functional group and orients the functional group in a favorable way within the binding-site region of the receptor [203-205]. A recent study , where MCSS approach was applied against the homology models of the ARs constructed by using the X-ray structure of rhodopsin as a template, successfully identified a few potent antagonists selective to the α1dAR.
It appears that if any homology model is constructed using a template of the X-ray GPCR in its apo state or inverse agonist bound type, the binding region needs be modified to accommodate various types of ligands [207-208]. For the purpose of rational drug design based upon a homology model of the CB1 receptor, it appears that the early intermediate stages of the active state of the receptor bound to an agonist, resembling the inactive state of the receptor, are sufficient as alternative structures to the fully activated structure for the receptor-based agonist design . In this case, it is crucial to identify the receptor residues involved in kinetically distinct steps in the receptor activation. For β2AR, it has been proposed that the agonist binding of β2AR occurs through at least three distinct steps [11,157]: 1) the first step involves an ionic/H-bond interaction between the protonated amine of the agonist and D1133.32 and N3127.39 of the receptor and aromatic stacking between the catechol ring of the ligand and F2906.52 of the receptor. A combination of these interactions composes the key initial ligand contacts to a minimal low-affinity receptor binding site; 2) the second step involves H-bonding interactions between the catechol hydroxyl groups of the ligand and S2035.42, S2045.43, and S2075.46 of the receptor . These interactions, which properly position the catechol ring of the ligand and H5, are required for the receptor to achieve the fully active state ; and 3) the third step involves a rotamer toggle switch of W2866.48/F2906.52 , which modulates H6 movement about the P6.50 kink, and H-bond interaction between the side chain hydroxyl group of the ligand and N2936.55 of the receptor. Accordingly, de Graaf and Rognan  modified the rotameric states of S2045.43 and S2075.46 of the X-ray of β2AR and successfully screened selective full and partial agonists.
Thus, similar steps of agonist binding can be considered for the CB1 receptor. Several issues need to be addressed: First, with a variety of ligands with distinct structures, the cannabinoid ligand binding pocket has not been well established. It is generally accepted that structurally diverse cannabinoid agonists bind to the binding sites uniquely defined for the individual classes of the ligand but with partial overlap  (see Fig. (3)). Second, in contrast to β2AR agonists, classical and non-classical cannabinoid agonists do not contain the protonated amine, while WIN55212-2 contains the morpholino N that can be protonated. On the other hand, similar to β2AR agonists, most cannabinoid agonists do contain at least one aromatic moiety. Thus, aromatic residues [95,96,107,120] in the extracellular side of the CB1 receptor would serve as the key initial ligand contacts to a minimal low-affinity receptor binding site. Third, it is conceivable that several aromatic residues on H5 of the CB1 receptor, including Y2755.39  for CP55940 and Y2755.39  and W2795.43  for WIN55212-2, perform a similar role as the agonist-contact residues S2035.42, S2045.43 and S2075.46  on H5 of β2AR. These residues need to be repositioned with a rotameric change for a full activation of the receptor . Fourth, the toggle switch W3566.48/F2003.36  and the VxxI helical groove  just proximal to the CWxP sequence of H6 can be the key interaction sites for the agonist binding contacts that induce the agonist-initiated receptor micro-conformational change [131,173].
Based upon the results from CB1 receptor mutagenesis, pharmacological and computational studies, it is possible to generate a CB1 receptor model in an early active state by modifying the rotameric states of the binding site residues. Candidate modifications include C3556.47 for CP55940  and F2003.36 [95,120]. These residues are known to be involved in receptor activation as well as ligand binding. Such early active state CB1 receptor models are useful in screening CB1 receptor selective full and partial agonists, as similarly demonstrated for β2AR . As one example of this strategy, Salo et al.  modified a homology model of the CB2 receptor and docked HU210 in the binding pocket prior to database screening to identify novel agonist candidates.
The CB1 receptor is a GPCR with conformational flexibility as indicated by its high basal activity. The CB1 receptor functional residues reported from mutational studies are utilized to describe the molecular events of the ligand-specific CB1 receptor activation from ligand binding to G protein signaling. For the CB1 receptor, as shown in many GPCRs, the integrity of the extracellular loops in ligand binding and coupling to the TM helical domain, the interference of the CWxP motif and the breakage of the ionic lock are important for receptor activation. Yet, little is known about the molecular structure of the CB1 receptor in the active state. But, it is possible to construct homology models of the CB1 receptor corresponding to the early stage of its activation that are suitable for identifying new therapeutic agents by applying the validated structure-based approaches, such as virtual HTS and FBA.
Support from NIH NIDA 5K01DA020663 and NCSA under MCB080037N is acknowledged. Drs. L. Padgett and A. Howlett are greatly acknowledged for their careful reading of the manuscript. Drs. L. Pedersen and L. Perera are acknowledged for their insightful discussion and suggestions.