G protein-coupled receptors: the evolution of structural insight

Abstract

G protein-coupled receptors (GPCR) comprise a diverse superfamily of over 800 proteins that have gained relevance as biological targets for pharmaceutical drug design. Although these receptors have been investigated for decades, three-dimensional structures of GPCR have only recently become available. In this review, we focus on the technological advancements that have facilitated efforts to gain insights into GPCR structure. Progress in these efforts began with the initial crystal structure determination of rhodopsin (PDB: 1F88) in 2000 and has continued to the most recently published structure of the A_1AR (PDB: 5UEN) in 2017. Numerous experimental developments over the past two decades have opened the door for widespread GPCR structural characterization. These efforts have resulted in the determination of three-dimensional structures for over 40 individual GPCR family members. Herein we present a comprehensive list and comparative analysis of over 180 individual GPCR structures. This includes a summary of different GPCR functional states crystallized with agonists, dual agonists, partial agonists, inverse agonists, antagonists, and allosteric modulators.

Introduction

G protein-coupled receptors (GPCR) comprise a superfamily of over 800 proteins that are the largest family of cell surface receptors in the human genome [1–3]. These proteins share a characteristic seven transmembrane spanning, alpha-helical structure [4]. The GPCR superfamily is commonly subdivided, based on sequence comparisons, into five distinct families: Rhodopsin (class A), Adhesion (class B), Secretin (class B), Glutamate (class C), and Frizzled/Taste2 (class F) [3, 5]. More details on the sequence-based analyses that led to these phylogenetic divisions are further discussed in the section titled “Phylogenetic classification/structure” in this review. GPCR have been implicated in numerous biological processes such as cognitive responses [6], cardiovascular functions [7], and cancer growth and development [8]. GPCR implication in human disease is reflected by the estimated 50% of pharmaceutical drugs that interact with these receptors [9].

GPCR mediate signal transduction cascades initiated by numerous extracellular molecules through which they produce downstream physiological responses [4, 8, 10]. However, receptor activity is not solely stimulated by binding of extracellular ligands. Constitutively active basal signaling [11] has been demonstrated in over 60 wild-type GPCR, including ADRB₂; A_2AR; and CB₁ [12]. Some GPCR, such as taste receptors [13, 14], adopt active state conformations upon interaction with other receptors [15]. Further details regarding GPCR activation can be found in the “GPCR function” section.

Although these receptors have diverse roles in cellular signaling and in physiology and pathophysiology, they share similar structural topology. This topology includes common structural features shown in figure 1 that include extracellular loops (EL1-3) and intracellular loops (IL1-3) that alternately connect a characteristic seven 251660288251659264 transmembrane (7TM) α-helical bundle (figure 1B) [3, 4]. Crystallographic approaches described in more detail in the section titled “Structural characterization” have revealed structural similarities shared by many GPCR found mainly within the 7TM domain. This structural homology, described in more detail in later sections titled by class, suggests similar means for cell signaling. Although GPCR have a shared topology and 7TM structure, there is diversity within the superfamily in regards to sequence composition and length, producing structural variations that are associated with functional specificity in regards to ligand binding or G protein coupling for different types of GPCR. Select structural differences are also described in more detail in later sections titled by class.

Figure 1.

(A) Topological cartoon representing the N-terminal domain, EL 1–3, 7TM domain in cylinders, IL1-3, and C-terminal domain. (B) Ribbon structure of rhodopsin (PDB: 1F88) demonstrating the arrangement of the 7TM α-helical domains within the characteristic membrane-spanning bundle. Here the helices are colored blue starting at the N-terminus fading to red at the C-terminus.

GPCR function

Although this review focuses on GPCR structure and structural characterization, GPCR play a significant role in signal transduction cascades that warrants a generalized overview of their functional and regulatory mechanisms. GPCR are a critical mediator in overall cell signaling, both through ligand-dependent and ligand-independent mechanisms. The majority of these receptors relay signals initiated by binding of extracellular ligands. These ligands are either naturally produced (endogenous) or externally administered (exogenous). Ligands are classified as agonists, inverse agonists, or antagonists. Agonists bind to the receptors and induce a conformational change to an active state, which increases signaling effects. Inverse agonists shift the receptor conformational equilibrium toward inactive conformations, thereby inhibiting basal activity. Antagonists, on the other hand, prevent binding of agonists or inverse agonists without affecting the dynamic conformational equilibrium, which prevents agonist-dependent receptor activation [16]. Changes in conformation within the TM domain, in turn, initiate a conformational change in the intracellular region. IL2 and IL3 have been found to contain critical interaction sites for G proteins or other cytoplasmic effectors [17]. This is illustrated in the crystal structure of ADRB₂-Gα_s complex (PDB: 3SN6). Residues located in IL2, TM5, and TM6 of ADRB₂ demonstrate an interaction interface with the α4 helix, α5-helix, αN-β1 junction, and β3 strand of Gα_s [18]. In the classical understanding of GPCR signaling, agonist binding promotes the formation of the activated receptor-G protein complex that modulates functional effects. A detailed analysis of G protein structure, regulation, and their involvement in signaling has been effectively summarized by Gilman [19]. The heterotrimeric G protein complex is made up of α, β, and γ subunits where the α subunit falls into four subfamilies: Gα_s, which stimulates adenylyl cyclase activity; Gα_i, which inhibits adenylyl cyclase activity; Gα_q, which activates phospholipase Cβ; and Gα_12/13, which promotes Rho activation. An activated receptor can interact with heterotrimeric G protein complexes. Receptors function as guanine nucleotide exchange factors (GEF) to promote dissociation of GDP from the Gα subunit in exchange for GTP (figure 2A) [4]. In the activated heterotrimeric G protein complex, the Gα-GTP bound subunit dissociates from the βγ subunit resulting in propagation of signaling cascades. However, subsequent studies have shown some receptors are able to reach an activated state through the formation of dimers and oligomers without agonist binding [4, 16]. Alternatively, GPCR activation can occur through interactions with adaptor and scaffolding proteins. Through specific binding sites, adaptor/scaffolding proteins, such as A-kinase anchoring proteins (AKAP) and β-arrestins (figure 2B), mediate interactions between the receptor and downstream second messengers by scaffolding a network of proteins which then operate as a large molecular complex [4]. Furthermore, receptors including the cannabinoid receptor 1 (CB₁) exhibit high basal signaling, independent of agonist activation [20]. High basal signaling might be indicative of conformational flexibility [21]; however, a study with a constitutively active ADRB₂ mutant has linked conformational flexibility with structural instability [11].

Figure 2.

(A) A classical example of GPCR signaling pathway. Receptors, such as ADRB₂, are in an inactive state until an agonist binds to activate the receptor. The activated receptor-G protein complex is formed resulting in GDP to GTP exchange by the G protein complex followed by the dissociation of the Gα subunit from the Gβγ dimer. Both Gα and Gβγ subunits, in turn, activate downstream effectors. Gα_x represents the general Gα subunit followed by diagrams for Gα_12/13, Gα_i, Gα_s, and Gα_q pathways. (B) β-arrestins can function as adaptor/scaffolding molecules activating the ERK (MAPK) cascade. GRK phosphorylates the activated receptor recruiting β-arrestin to the phosphorylation sites along with Raf, ERK (MAPK), and MEK. This association triggers activation of the ERK (MAPK) cascade leading to cytosolic signaling pathways. Figures 2A and 2B were adapted from Pierce, et al. [4].

Receptor signaling is modulated by various mechanisms to control cellular functions. Three such processes are highlighted here - deactivation, desensitization, and receptor internalization. At the receptor level, members of the RGS family of proteins serve as GPTase activating proteins (GAPs) and accelerate deactivation by enhancing the rate of Gα-catalyzed hydrolysis of GTP to GDP by factors up to 2000-fold (figure 3A) [22]. In other cases of continuous agonist stimulation, the receptor can be phosphorylated by downstream second-messenger protein kinases or a family of G protein-coupled receptor kinases (GRK). Following phosphorylation, β-arrestin is recruited to the receptor leading to the inhibition of receptor-G protein interaction via steric hindrance. The 251659264 receptor-β-arrestin complex is targeted and removed from the cell surface through endocytosis. The receptor is then either degraded or recycled back to the cell surface in the inactive conformation (figure 3B). Receptor internalization is mediated by β-arrestin, clathrin-coated pits, and caveolae through diverse mechanisms [23]. Further in-depth evaluations of GPCR function can be found in excellent reviews by Pierce, et al. [4] and Syrovatkina, et al. [16].

Figure 3.

(A) Receptor deactivation is mediated by the RGS family of proteins. RGS alters the conformation of Gα-GTP complex making it a better hydrolase, which accelerates the hydrolysis of GTP to GDP. (B) GPCR desensitization through internalization occurs when GRK phosphorylates the activated receptor promoting β-arresting binding, which sterically hinders receptor-G protein interaction. The receptor is either degraded or recycled back to the cell surface. Figure 3A was adapted from Neubig, et al. [191] and 3B was adapted from Pierce, et al. [4].

Structural characterization

Due to its high endogenous concentration, the GPCR rhodopsin was purified from bovine rod outer segment (ROS) membranes [24] leading to its crystallization in 2000 (figure 4; PDB: 1F88) [25]. To date, rhodopsin is the only GPCR that has been purified from its natural source for structural studies. The next GPCR successfully crystallized was ADRB₂ (PDB: 2RH1 [26], 2R4R/2R4S [27]) seven years later. This delay was due to a need for numerous technological advances required to express, purify and crystallize these lipid-soluble targets. The most critical of these advances were improved lipid phases [28–30], lipidic cubic phase (LCP) methods for crystallization [31], and the incorporation of soluble fusion partners [31, 32].

Figure 4.

Timeline of availability for individual representative GPCR crystal structure structures including protein fusion partners. Rho (1F88), ADRB₂ (2R4R), ADRB₁ (2VT4), A_2AR (3EML), TACR₁ (2KS9), CXCR4 (3ODU), D₃R (3PBL), H₁R (3RZE), S1P₁ (3V2W), M₂R (3UON), CXCR1 (2LNL), M₃R (4DAJ), κ-OR (4DJH), μ-OR (4DKL), NOP (4EA3), δ-OR (4EJ4), PAR1 (3VW7), NSTR₁ (3ZEV), 5-HT_1B (4IAQ), 5-HT_2B (4IB4), SMO (4JKV), CRF₁R (4K5Y), CCR5 (4MBS) P2Y₁₂ (4NTJ), mGlu₅ (4OO9), mGlu₁ (4OR2), FFAR₁ (4PHU), OX₂ (4S0V), P2Y₁ (4XNW), HHV-5 US28 (4XT1), AT₁R (4YAY), LPA₁ (4Z34), OX₁ (4ZJ8), M₁R (5CXV), M₄R (5DSG), GCGR (5EE7), CCR9 (5LWE), ET-B (5GLH), CCR2 (5T1A), CB₁ (5TGZ), A_1AR (5UEN), AT₂R (5UNF).

Purifying hydrophobic membrane proteins requires additional steps compared to water-soluble proteins. Prior to purification, membrane proteins must be removed from their native lipid environment. Although solubilizing detergents are required to enable efficient extraction of GPCR from the membranes, it is essential to select a mild detergent that discourages protein denaturation. It is important to note that detergent protocols used for one receptor may not be appropriate to other receptors, thus making detergent screening a critical step in the obtaining viable protein. Rhodopsin (PDB: 1F88) was effectively solubilized in a mixed micellar solution containing heptane-1,2,3-triol (HPTO), nonyl glucoside, and zwitterionic lauryldimethylamine-N-oxide (LDAO) [24]. Both ADRB₂ structures from 2007 (PDB: 2RH1 [26], 2R4R/2R4S [27]) were solubilized in dodecylmaltoside. Often, detergents used for extraction are not sufficient to stabilize protein and a detergent exchange is required [31]. This method was observed in the crystallization trials of the ADRB₂-Gs complex (PDB: 3SN6) where the complex was formed in dodecylmaltoside solution and exchanged into maltose neopentylglycerol detergent (MNG-3) [18].

Crystallization of membrane proteins has been accomplished through the use of different lipid phases including micelles, bicelles, and nanodiscs. These different lipid phases allow for protein stabilization and promote crystal lattice formation [28–30]. Detergent micelles surround the hydrophobic GPCR structural regions and allow crystal lattice contacts to form between exposed protein loop structures [28]. Lipid-based bicelles and nanodiscs, on the other hand, act as lipidic mimics of a native bilayer environment. Bicelles are formed via the combination of a detergent or short-chain lipid with a long chain lipid, such as dimyristoyl phosphatidylcholine (DMPC) [29]. A nanodisc is a non-covalently assembled phospholipid bilayer encircled by membrane scaffold proteins, such as plasma lipoproteins [33]. Rhodopsin (PDB: 1F88 [24]) and ADRB₂ (PDB: 2R4R/2R4S [27]) were crystallized in micelles and DMPC/CHAPSO bicelles, respectively. Nanodiscs were used in the crystallization of the nanobody-stabilized ADRB₂ (PDB: 3P0G).

Following many detergent optimization efforts, rhodopsin was crystallized by vapor diffusion [24]. Vapor diffusion forces protein solutions to reach supersaturation prior to crystallization. In this method, protein solutions are mixed with a crystallization solution and positioned in a sitting-drop or hanging-drop orientation in an airtight chamber that also contains the crystallization solution. The chemical equilibrium between the drop and the chamber results in the evaporation of volatile species, which diffuse from the drop to the well. This diffusion leads to a slow saturation of protein within the drop allowing the protein-detergent complexes to form crystal lattice contacts with neighboring molecules [34]. In lipidic cubic phase (LCP) methods, proteins are placed in a membrane-like environment where they can diffuse and interact with each other to form crystal lattice contacts on both complementary hydrophobic and hydrophilic regions [31]. Crystals of rhodopsin (PDB: 1F88 [25]) and ADRB₂ (PDB: 2R4R/2R4S [27]) were grown by hanging drop diffusion, whereas ADRB₂ (PRB: 2RH1 [26]) and ADRB₂-Gs complex (PDB: 3SN6 [18]) crystallization experiments implemented LCP methods.

In determining crystallographic structures, molecular replacement has been successfully utilized to help in determining the phase of an unknown target by applying the phase of a closely related and previously characterized target. This approach was implemented in the analysis of the ADRB₂ structure [26]. Multi-wavelength anomalous scattering (MAD) and single-wavelength anomalous scattering (SAD) methods have impacted atomic-level structure determinations of GPCR when a heavy atom anomalous scatterer is incorporated into the protein structure. Structural determination using MAD phasing was implemented in the first crystal structure of rhodopsin (PDB: 1F88) [25].

Developments in protein engineering and heterologous protein expression have accelerated structural determinations of GPCR. Site-directed mutagenesis has been beneficial for the stabilization of receptor structure, as well as determination of the functional activity of many GPCR [31]. Mutagenesis has also enabled the introduction of fusion partners into protein sequences. Fusion partners are designed to be highly stable, compact, and easily crystallized. These partners maintain essential surface contacts and increase protein solubility, making them suitable replacements for flexible domains [31, 32]. The position of the fusion partner and the number of residues conjoining the fusion partner to the protein may alter expression and/or stability; nevertheless, optimizing the number of linker residues can minimize adverse effects. A Fab5 epitope was engineered into IL3 of ADRB₂ (PDB: 2R4R/2R4S) by mutagenesis, which allowed a Fab5 monoclonal antibody to bind to the epitope [27]. Fab5 stabilized the receptor conformation and increased the polar surface area necessary for crystal lattice contacts. Since the initial use of the Fab5 antibody, many GPCR have been successfully engineered with fusion partners accelerating the number solved of crystallographic structures in the last two decades as illustrated in the timeline in figure 4. T4 lysozyme has been the most widely used fusion partner in multiple receptors such as adenosine A_2A receptor (A_2AR; PDB: 3EML) [37] and metabotropic glutamate receptor 5 (mGlu₅; PDB: 4OO9) [38]. Thermostabilized apocytochrome b₅₆₂RIL (BRIL) is a highly versatile fusion partner that has been appended to the N-terminal domain of nociception receptor 1 (NOP; PDB: 4EA3) [39] and C-terminal domain of smoothened receptor (SMO; PDB: 4JKV) [40]. Pyrococcus abyssi glycogen synthase (PGS) and rubredoxin emerged as fusion partners later in the timeline of GPCR characterization. These tools have been successfully used in analysis of the orexin 1 (OX₁; PDB: 4ZJ8) [41] and purinergic receptor 1 (P2Y₁; PDB: 4XNW) receptors [42]. Although the addition of fusion protein sequences have been effective in the analysis of many GPCR, it is important to note that engineering designs are often specific for one protein and require optimization for each application to new receptor sequences.

GPCR loops and termini, which are characteristically flexible and difficult to crystallize, can result in additional challenges. Proteins truncated or modified at these areas can reduce flexibility. However, simple truncations can also reduce the polar surface area of the protein, which is a characteristic crucial for forming the crystal lattice contacts required for crystal formation. Rhodopsin has a relatively short IL3 with ~3 amino acids and C-terminal domain with ~25 amino acids, in contrast, ADRB₂ has a lengthy IL3 with ~28 amino acids and a C-terminal domain ~70 amino acids [43]. Two early structures of ADRB₂ were engineered differently in these regions to achieve crystal lattice formation during crystallization. In the ADRB₁-Fab5 complex (PDB: 2R4R/2R4S), truncating the final 48 amino acids in the unstructured C-terminal domain optimized crystal size and uniformity [27]. In another structure of ADRB₂ (PDB: 2RH1), IL3 was completely removed and replaced with the soluble fusion partner, T4 lysozyme, which promoted the growth of diffraction quality crystals [26]. Amino acid truncations have also been found to have a variable effect on protein expression. Truncations at the N-terminus reduced expression. However, replacing the N-terminus with BRIL maintained similar expression as constructs with the complete N-terminus [39]. This was the case with NOP (PDB: 4EA3) where an N-terminal replacement with BRIL and a 31 amino acid deletion at the C-terminus significantly increased protein expression [31, 39].

There have been challenges in GPCR structure determination due to their hydrophobic nature and instability outside of the hydrophobic membrane environment [44, 45]. Since the first complete x-ray crystallographic structure of a GPCR (bovine rhodopsin; PDB: 1F88) was solved in 2000 [25], there are now over 180 comprehensive structures of GPCR in the Protein Data Bank (www.rcsb.org [46]) listed in table 1. More than 150 of these structures co-crystallized with ligands are described in more detail in table 2. However, less than 45 distinct family members are represented out of a superfamily of over 800 proteins. This limitation in representation suggests that significantly more work is yet to be done.

Table 1.

A comprehensive list of GPCR crystal structures according to date deposited into the Protein Data Bank as of April 19, 2017.

GPCR Crystal Structures
Individual Receptors	Family	Class/Subclass	Species	Receptor	PDB [46] ID	Resolution (Å)	Date Deposited
1	Rhodopsin	Class Aα	Bovine	Rho	1F88 [25]	2.80	6/29/00
2	Rhodopsin	Class Aα	Bovine	Rho	1HZX [94]	2.80	1/26/01
3	Rhodopsin	Class Aα	Bovine	Rho	1JFP [95]	Solution NMR	6/21/01
4	Rhodopsin	Class Aα	Bovine	Rho	1L9H [96]	2.60	3/23/02
5	Rhodopsin	Class Aα	Bovine	Rho	1LN6 [97]	Solution NMR	5/3/02
6	Rhodopsin	Class Aα	Bovine	Rho	1GZM [98]	2.65	5/24/02
7	Rhodopsin	Class Aα	Bovine	Rho	1U19 [99]	2.20	7/15/04
8	Rhodopsin	Class Aα	Bovine	Rho	2G87 [100]	2.60	3/2/06
9	Rhodopsin	Class Aα	Bovine	Rho	2HPY [101]	2.80	7/18/06
10	Rhodopsin	Class Aα	Bovine	Rho	2I35 [102]	3.80	8/17/06
11	Rhodopsin	Class Aα	Bovine	Rho	2I36 [102]	4.10	8/17/06
12	Rhodopsin	Class Aα	Bovine	Rho	2I37 [102]	4.15	8/17/06
13	Rhodopsin	Class Aα	Bovine	Rho	2J4Y [103]	3.40	9/7/06
14	Rhodopsin	Class Aα	Bovine	Rho	2PED [104]	2.95	4/2/07
15	Rhodopsin	Class Aα	Bovine	Rho	3C9L [105]	2.65	2/16/08
16	Rhodopsin	Class Aα	Bovine	Rho	3C9M [105]	3.40	2/16/08
17	Rhodopsin	Class Aα	Bovine	Rho	3CAP [73]	2.90	2/20/08
18	Rhodopsin	Class Aα	Bovine	Rho	3DQB [106]	3.20	7/9/08
19	Rhodopsin	Class Aα	Bovine	Rho	2X72 [107]	3.00	2/22/10
20	Rhodopsin	Class Aα	Bovine	Rho	3OAX[108]	2.60	8/5/10
21	Rhodopsin	Class Aα	Bovine	Rho	3PQR [109]	2.85	11/26/10
22	Rhodopsin	Class Aα	Bovine	Rho	3PXO [109]	3.00	12/10/10
23	Rhodopsin	Class Aα	Bovine	Rho	4A4M [110]	3.30	10/17/11
24	Rhodopsin	Class Aα	Bovine	Rho	4J4Q [111]	2.65	2/7/13
25	Rhodopsin	Class Aα	Bovine	Rho	4BEY [112]	2.90	3/12/13
26	Rhodopsin	Class Aα	Bovine	Rho	4BEZ [112]	3.30	3/12/13
27	Rhodopsin	Class Aα	Bovine	Rho	4PXF [113]	2.75	3/23/14
28	Rhodopsin	Class Aα	Bovine	Rho	4X1H [114]	2.29	11/24/14
29	Rhodopsin	Class Aα	Human	Rho	4ZWJ [115]	3.30	5/19/15
30	Rhodopsin	Class Aα	Bovine	Rho	5DYS [116]	2.30	9/25/15
31	Rhodopsin	Class Aα	Bovine	Rho	5EN0 [116]	2.81	11/8/15
32	Rhodopsin	Class Aα	Bovine	Rho	5TE3 [117]	2.70	9/20/16
33	Rhodopsin	Class Aα	Bovine	Rho	5TE5 [117]	4.01	9/21/16
34	Rhodopsin	Class Aα	Turkey	ADRB1	2VT4 [72]	2.70	5/9/08
35	Rhodopsin	Class Aα	Turkey	ADRB1	2Y00 [118]	2.50	11/30/10
36	Rhodopsin	Class Aα	Turkey	ADRB1	2Y01[118]	2.60	11/30/10
37	Rhodopsin	Class Aα	Turkey	ADRB1	2Y02 [118]	2.60	11/30/10
38	Rhodopsin	Class Aα	Turkey	ADRB1	2Y03 [118]	2.85	11/30/10
39	Rhodopsin	Class Aα	Turkey	ADRB1	2Y04 [118]	3.05	11/30/10
40	Rhodopsin	Class Aα	Turkey	ADRB1	2YCW [74]	3.00	3/17/11
41	Rhodopsin	Class Aα	Turkey	ADRB1	2YCX [74]	3.25	3/17/11
42	Rhodopsin	Class Aα	Turkey	ADRB1	2YCY [74]	3.15	3/17/11
43	Rhodopsin	Class Aα	Turkey	ADRB1	2YCZ [74]	3.65	3/17/11
44	Rhodopsin	Class Aα	Turkey	ADRB1	3ZPQ [119]	2.80	3/1/13
45	Rhodopsin	Class Aα	Turkey	ADRB1	3ZPR [119]	2.70	3/1/13
46	Rhodopsin	Class Aα	Turkey	ADRB1	4AMI [120]	3.20	3/11/12
47	Rhodopsin	Class Aα	Turkey	ADRB1	4AMJ [120]	2.30	3/12/12
48	Rhodopsin	Class Aα	Turkey	ADRB1	4BVN [69]	2.10	6/26/13
49	Rhodopsin	Class Aα	Turkey	ADRB1	4GPO [121]	3.50	8/21/12
50	Rhodopsin	Class Aα	Turkey	ADRB1	5A8E [122]	2.40	7/15/15
51	Rhodopsin	Class Aα	Turkey	ADRB1	5F8U [123]	3.35	12/9/15
52	Rhodopsin	Class Aα	Human	ADRB2	2R4R [27]	3.40	8/31/07
53	Rhodopsin	Class Aα	Human	ADRB2	2R4S [27]	3.40	8/31/07
54	Rhodopsin	Class Aα	Human	ADRB2	2RH1 [26]	2.40	10/5/07
55	Rhodopsin	Class Aα	Human	ADRB2	3D4S [124]	2.80	5/14/08
56	Rhodopsin	Class Aα	Human	ADRB2	3KJ6 [125]	3.40	11/2/09
57	Rhodopsin	Class Aα	Human	ADRB2	3NY8 [126]	2.84	7/14/10
58	Rhodopsin	Class Aα	Human	ADRB2	3NY9 [126]	2.84	7/14/10
59	Rhodopsin	Class Aα	Human	ADRB2	3NYA [126]	3.16	7/14/10
60	Rhodopsin	Class Aα	Human	ADRB2	3P0G [127]	3.50	9/28/10
61	Rhodopsin	Class Aα	Human	ADRB2	3PDS [128]	3.50	10/24/10
62	Rhodopsin	Class Aα	Human	ADRB2	3SN6 [18]	3.20	6/28/11
63	Rhodopsin	Class Aα	Human	ADRB2	4GBR [129]	3.99	7/27/12
64	Rhodopsin	Class Aα	Human	ADRB2	4LDE [130]	2.79	6/24/13
65	Rhodopsin	Class Aα	Human	ADRB2	4LDL [130]	3.10	6/24/13
66	Rhodopsin	Class Aα	Human	ADRB2	4LDO [130]	3.20	6/24/13
67	Rhodopsin	Class Aα	Human	ADRB2	4QKX [131]	3.30	6/10/14
68	Rhodopsin	Class Aα	Human	ADRB2	5D5A [132]	2.48	8/10/15
69	Rhodopsin	Class Aα	Human	ADRB2	5D5B [132]	3.80	8/10/15
70	Rhodopsin	Class Aα	Human	ADRB2	5D6L [133]	3.20	8/12/15
71	Rhodopsin	Class Aα	Human	ADRB2	5JQH [134]	3.20	5/5/16
72	Rhodopsin	Class Aα	Human	H1R	3RZE [135]	3.10	5/11/11
73	Rhodopsin	Class Aα	Human	D3R	3PBL [6]	2.89	10/20/10
74	Rhodopsin	Class Aα	Human	5-HT1B	4IAQ [136]	2.80	12/7/12
75	Rhodopsin	Class Aα	Human	5-HT1B	4IAR [136]	2.70	12/7/12
76	Rhodopsin	Class Aα	Human	5-HT2B	4IB4 [137]	2.70	12/7/12
77	Rhodopsin	Class Aα	Human	5-HT2B	4NC3 [138]	2.80	10/23/13
78	Rhodopsin	Class Aα	Human	5-HT2B	5TVN [139]	2.90	11/9/16
79	Rhodopsin	Class Aα	Human	M1R	5CXV [140]	2.70	7/29/15
80	Rhodopsin	Class Aα	Human	M2R	3UON [141]	3.00	11/16/11
81	Rhodopsin	Class Aα	Human	M2R	4MQS [142]	3.50	9/16/13
82	Rhodopsin	Class Aα	Human	M2R	4MQT [142]	3.70	9/16/13
83	Rhodopsin	Class Aα	Rat	M3R	4DAJ [143]	3.40	1/12/12
84	Rhodopsin	Class Aα	Rat	M3R	4U14 [144]	3.57	7/15/14
85	Rhodopsin	Class Aα	Rat	M3R	4U15 [144]	2.80	7/15/14
86	Rhodopsin	Class Aα	Rat	M3R	4U16 [144]	3.70	7/15/14
87	Rhodopsin	Class Aα	Human	M4R	5DSG [140]	2.60	9/17/15
88	Rhodopsin	Class Aα	Human	A1AR	5UEN [145]	3.20	1/3/17
89	Rhodopsin	Class Aα	Human	A2AR	3EML [37]	2.60	9/24/08
90	Rhodopsin	Class Aα	Human	A2AR	3PWH [146]	3.30	12/8/10
91	Rhodopsin	Class Aα	Human	A2AR	3QAK [68]	2.71	1/11/11
92	Rhodopsin	Class Aα	Human	A2AR	2YDO [147]	3.00	3/23/11
93	Rhodopsin	Class Aα	Human	A2AR	2YDV [147]	2.60	3/24/11
94	Rhodopsin	Class Aα	Human	A2AR	3REY [146]	3.31	4/5/11
95	Rhodopsin	Class Aα	Human	A2AR	3RFM [146]	3.60	4/6/11
96	Rhodopsin	Class Aα	Human	A2AR	3VG9 [148]	2.70	8/4/11
97	Rhodopsin	Class Aα	Human	A2AR	3VGA [148]	3.10	8/4/11
98	Rhodopsin	Class Aα	Human	A2AR	3UZA [149]	3.27	12/7/11
99	Rhodopsin	Class Aα	Human	A2AR	3UZC [149]	3.34	12/7/11
100	Rhodopsin	Class Aα	Human	A2AR	4EIY [66]	1.80	4/6/12
101	Rhodopsin	Class Aα	Human	A2AR	4UG2 [150]	2.60	3/21/15
102	Rhodopsin	Class Aα	Human	A2AR	4UHR [150]	2.60	3/25/15
103	Rhodopsin	Class Aα	Human	A2AR	5IU4 [151]	1.72	3/17/16
104	Rhodopsin	Class Aα	Human	A2AR	5IU7 [151]	1.90	3/17/16
105	Rhodopsin	Class Aα	Human	A2AR	5IU8 [151]	2.00	3/17/16
106	Rhodopsin	Class Aα	Human	A2AR	5IUA [151]	2.20	3/17/16
107	Rhodopsin	Class Aα	Human	A2AR	5IUB [151]	2.10	3/17/16
108	Rhodopsin	Class Aα	Human	A2AR	5K2A [152]	2.50	5/18/16
109	Rhodopsin	Class Aα	Human	A2AR	5K2B [152]	2.50	5/18/16
110	Rhodopsin	Class Aα	Human	A2AR	5K2C [152]	1.90	5/18/16
111	Rhodopsin	Class Aα	Human	A2AR	5K2D [152]	1.90	5/18/16
112	Rhodopsin	Class Aα	Human	A2AR	5G53 [153]	3.40	5/19/16
113	Rhodopsin	Class Aα	Human	A2AR	5UIG [154]	3.50	1/13/17
114	Rhodopsin	Class Aα	Human	S1P1	3V2W [155]	3.35	12/12/11
115	Rhodopsin	Class Aα	Human	S1P1	3V2Y [155]	2.80	12/12/11
116	Rhodopsin	Class Aα	Human	LPA1	4Z34 [156]	3.00	3/30/15
117	Rhodopsin	Class Aα	Human	LPA1	4Z35 [156]	2.90	3/30/15
118	Rhodopsin	Class Aα	Human	LPA1	4Z36 [156]	2.90	3/30/15
119	Rhodopsin	Class Aα	Human	CB1	5TGZ [157]	2.80	9/28/16
120	Rhodopsin	Class Aα	Human	CB1	5U09 [158]	2.60	11/23/16
121	Rhodopsin	Class Aβ	Rat	NTSR1	4GRV [159]	2.80	8/27/12
122	Rhodopsin	Class Aβ	Rat	NTSR1	3ZEV [160]	3.00	12/7/12
123	Rhodopsin	Class Aβ	Rat	NTSR1	4BUO [160]	2.75	6/21/13
124	Rhodopsin	Class Aβ	Rat	NTSR1	4BV0 [160]	3.10	6/24/13
125	Rhodopsin	Class Aβ	Rat	NTSR1	4BWB [160]	3.57	7/1/13
126	Rhodopsin	Class Aβ	Rat	NTSR1	4XEE [161]	2.90	12/23/14
127	Rhodopsin	Class Aβ	Rat	NTSR1	4XES [161]	2.60	12/24/14
128	Rhodopsin	Class Aβ	Rat	NTSR1	5T04 [162]	3.30	8/16/16
129	Rhodopsin	Class Aβ	Human	OX1	4ZJ8 [41]	2.75	4/29/15
130	Rhodopsin	Class Aβ	Human	OX1	4ZJC [41]	2.83	4/29/15
131	Rhodopsin	Class Aβ	Human	OX2	4S0V [163]	2.50	1/6/15
132	Rhodopsin	Class Aβ	Human	TACR1	2KS9 [164]	Solution NMR	12/31/09
133	Rhodopsin	Class Aβ	Human	TACR1	2KSA [164]	Solution NMR	12/31/09
134	Rhodopsin	Class Aβ	Human	TACR1	2KSB [164]	Solution NMR	12/31/09
135	Rhodopsin	Class Aβ	Human	ET-B	5GLI [165]	2.5	7/11/16
136	Rhodopsin	Class Aβ	Human	ET-B	5GLH [165]	2.8	7/11/16
137	Rhodopsin	Class Aγ	Human	CXCR1	2LNL [166]	Solid-State NMR	12/31/11
138	Rhodopsin	Class Aγ	Human	CCR2	5T1A [167]	2.81	8/18/16
139	Rhodopsin	Class Aγ	Human	CXCR4	3ODU [168]	2.50	8/11/10
140	Rhodopsin	Class Aγ	Human	CXCR4	3OE0 [168]	2.90	8/12/10
141	Rhodopsin	Class Aγ	Human	CXCR4	3OE6 [168]	3.20	8/12/10
142	Rhodopsin	Class Aγ	Human	CXCR4	3OE8 [168]	3.10	8/12/10
143	Rhodopsin	Class Aγ	Human	CXCR4	3OE9 [168]	3.10	8/12/10
144	Rhodopsin	Class Aγ	Human	CXCR4	4RWS [169]	3.10	12/5/14
145	Rhodopsin	Class Aγ	Human	CCR5	4MBS [170]	2.71	8/19/13
146	Rhodopsin	Class Aγ	Human	CCR9	5LWE [171]	2.80	9/16/16
147	Rhodopsin	Class Aγ	Human	NOP	4EA3 [39]	3.01	3/22/12
148	Rhodopsin	Class Aγ	Human	NOP	5DHG [172]	3.00	8/30/15
149	Rhodopsin	Class Aγ	Human	NOP	5DHH [172]	3.00	8/31/15
150	Rhodopsin	Class Aγ	Human	κ-OR	4DJH [173]	2.90	2/1/12
151	Rhodopsin	Class Aγ	Mouse	μ-OR	4DKL [174]	2.80	2/3/12
152	Rhodopsin	Class Aγ	Mouse	μ-OR	5C1M[175]	2.10	6/15/15
153	Rhodopsin	Class Aγ	Mouse	δ-OR	4EJ4 [176]	3.40	4/6/12
154	Rhodopsin	Class Aγ	Human	δ-OR	4N6H [70]	1.80	10/12/13
155	Rhodopsin	Class Aγ	Human	δ-OR	4RWA [177]	3.28	12/1/14
156	Rhodopsin	Class Aγ	Human	δ-OR	4RWD [177]	2.70	12/2/14
157	Rhodopsin	Class Aγ	Human	AT1R	4YAY [178]	2.90	2/18/15
158	Rhodopsin	Class Aγ	Human	AT1R	4ZUD [179]	2.80	5/15/15
159	Rhodopsin	Class Aγ	Human	AT2R	5UNF [180]	2.80	1/30/17
160	Rhodopsin	Class Aγ	Human	AT2R	5UNG [180]	2.80	1/30/17
161	Rhodopsin	Class Aγ	Human	AT2R	5UNH [180]	2.90	1/30/17
162	Rhodopsin	Class Aγ	HHV-5	HHV-5 US28	4XT1 [181]	2.89	1/22/15
163	Rhodopsin	Class Aγ	HHV-5	HHV-5 US28	4XT3 [181]	3.80	1/22/15
164	Rhodopsin	Class Aδ	Human	P2Y1	4XNW [182]	2.70	1/16/15
165	Rhodopsin	Class Aδ	Human	P2Y1	4XNV [182]	2.20	1/16/15
166	Rhodopsin	Class Aδ	Human	P2Y12	4NTJ [42]	2.62	12/2/13
167	Rhodopsin	Class Aδ	Human	P2Y12	4PXZ [183]	2.50	3/25/14
168	Rhodopsin	Class Aδ	Human	P2Y12	4PY0 [183]	3.10	3/25/14
169	Rhodopsin	Class Aδ	Human	PAR1	3VW7 [71]	2.20	8/7/12
170	Rhodopsin	Class Aδ	Human	FFAR1	4PHU [184]	2.33	5/7/14
171	Secretin	Class B	Human	GCGR	4L6R [185]	3.30	6/12/13
172	Secretin	Class B	Human	GCGR	5EE7 [78]	2.50	10/22/15
173	Secretin	Class B	Human	CRF1R	4K5Y [186]	2.98	4/15/13
174	Secretin	Class B	Human	CRF1R	4Z9G [187]	3.18	4/10/15
175	Glutamate	Class C	Human	mGlu1	4OR2 [188]	2.80	2/10/14
176	Glutamate	Class C	Human	mGlu5	4OO9 [38]	2.60	1/31/14
177	Glutamate	Class C	Human	mGlu5	5CGC [189]	3.10	7/9/15
178	Glutamate	Class C	Human	mGlu5	5CGD [189]	2.60	7/9/15
179	Frizzled/Taste2	Class F	Human	SMO	4JKV [40]	2.45	3/11/13
180	Frizzled/Taste2	Class F	Human	SMO	4N4W [190]	2.80	10/8/13
181	Frizzled/Taste2	Class F	Human	SMO	4O9R [131]	3.20	1/2/14
182	Frizzled/Taste2	Class F	Human	SMO	4QIM [190]	2.61	5/31/14
183	Frizzled/Taste2	Class F	Human	SMO	4QIN [190]	2.60	5/31/14
184	Frizzled/Taste2	Class F	Human	SMO	5L7D [93]	3.20	6/3/16
185	Frizzled/Taste2	Class F	Human	SMO	5L7I [93]	3.30	6/3/16

Table 2.

A comprehensive list of GPCR crystallized with ligands deposited into the Protein Data Bank as of April 19, 2017.

	PDB [46] ID	Class/Subclass	GPCR	Ligand Name	Ligand Type
1	1F88 [25]	Class Aα	Rho	11-cis-Retinal
2	1GZM [98]	Class Aα	Rho	11-cis-Retinal
3	1HZX [94]	Class Aα	Rho	11-cis-Retinal
4	1JFP [95]	Class Aα	Rho	all-trans-Retinal
5	1L9H [96]	Class Aα	Rho	11-cis-Retinal
6	1LN6 [97]	Class Aα	Rho	all-trans-Retinal
7	1U19 [99]	Class Aα	Rho	11-cis-Retinal
8	2G87 [100]	Class Aα	Rho	all-trans-Retinal
9	2HPY [101]	Class Aα	Rho	all-trans-Retinal
10	2I35 [102]	Class Aα	Rho	11-cis-Retinal
11	2J4Y [103]	Class Aα	Rho	11-cis-Retinal
12	2PED [104]	Class Aα	Rho	9-cis-Retinal
13	2X72 [107]	Class Aα	Rho	11-cis-Retinal
14	3C9L [105]	Class Aα	Rho	11-cis-Retinal
15	3C9M [105]	Class Aα	Rho	11-cis-Retinal
16	3OAX [108]	Class Aα	Rho	11-cis-Retinal
17	3PQR [109]	Class Aα	Rho	all-trans-Retinal
18	3PXO [109]	Class Aα	Rho	all-trans-Retinal
19	4A4M [110]	Class Aα	Rho	all-trans-Retinal
20	5DYS [116]	Class Aα	Rho	all-trans-Retinal
21	5EN0 [116]	Class Aα	Rho	all-trans-Retinal
22	5TE5 [117]	Class Aα	Rho	(2E)-{(4E)-4-[(3E)-4-(2,6,6-trimethylcyclohex- 1-en-1-yl)but-3-en-2-ylidene]cyclohex-2-en- 1-ylidene}acetaldehyde
23	4J4Q [111]	Class Aα	Rho	B-Octylglucoside	Agonist-stabilized active receptor conformation
24	2VT4 [72]	Class Aα	ADRB1	Cyanopindolol	Antagonist
25	2Y00 [118]	Class Aα	ADRB1	Dobutamine	Partial Agonist
26	2Y01 [118]	Class Aα	ADRB1	Dobutamine	Partial Agonist
27	2Y02 [118]	Class Aα	ADRB1	Carmoterol	Agonist
28	2Y03 [118]	Class Aα	ADRB1	Isoprenaline	Agonist
29	2Y04 [118]	Class Aα	ADRB1	Salbutamol	Partial Agonist
30	2YCW [74]	Class Aα	ADRB1	Carazolol	Antagonist
31	2YCX [74]	Class Aα	ADRB1	Cyanopindolol	Antagonist
32	2YCY [74]	Class Aα	ADRB1	Cyanopindolol	Antagonist
33	2YCZ [74]	Class Aα	ADRB1	Iodocyanopindolol	Antagonist
34	3ZPQ [119]	Class Aα	ADRB1	4-(Piperazin-1- yl)-1H-indole	Antagonist
35	3ZPR [119]	Class Aα	ADRB1	4-Methyl-2-(piperazin-1-yl)quinoline	Antagonist
36	4AMI [120]	Class Aα	ADRB1	Bucindolol	Agonist
37	4AMJ [120]	Class Aα	ADRB1	Carvedilol	Agonist
38	4BVN [69]	Class Aα	ADRB1	Cyanopindolol	Antagonist
39	5A8E [122]	Class Aα	ADRB1	7-methylcyanopindolol	Agonist
40	5F8U [123]	Class Aα	ADRB1	4-{[(2S)-3-(tert-butylamino)-2-hydroxypropyl]oxy}-3H-indole-2-carbonitrile	Antagonist
41	2RH1 [26]	Class Aα	ADRB2	Carazolol	Antagonist
42	3D4S [124]	Class Aα	ADRB2	Timolol maleate	Antagonist
43	3NY8 [126]	Class Aα	ADRB2	ICI 118,551	Antagonist
44	3NY9 [126]	Class Aα	ADRB2	Ethyl 4-({(2S)-2-hydroxy-3-[(1-methylethyl)amino]propyl}oxy)- 3-methyl-1-benzofuran-2-carboxylate	Antagonist
45	3NYA [126]	Class Aα	ADRB2	Alprenolol	Antagonist
46	3P0G [127]	Class Aα	ADRB2	BI 167107	Agonist
47	3PDS [128]	Class Aα	ADRB2	FAUC50	Agonist
48	3SN6 [18]	Class Aα	ADRB2	BI 167107	Agonist
49	4GBR [129]	Class Aα	ADRB2	BI 167107	Agonist
50	4LDE [130]	Class Aα	ADRB2	BI 167107	Agonist
51	4LDL [130]	Class Aα	ADRB2	Hydroxybenzyl isoproterenol	Agonist
52	4LDO [130]	Class Aα	ADRB2	Epinephrine	Agonist
53	4QKX [131]	Class Aα	ADRB2	FAUC37	Agonst
54	5D5A [132]	Class Aα	ADRB2	Carazolol	Antagonist
55	5D5B [132]	Class Aα	ADRB2	Carazolol	Antagonist
56	5D6L [133]	Class Aα	ADRB2	Carazolol	Antagonist
57	5JQH [134]	Class Aα	ADRB2	Carazolol	Antagonist
58	3RZE [135]	Class Aα	H1R	Doxepin (E/Z)	Antagonist
59	3PBL [6]	Class Aα	D3R	Eticlopride	Antagonist
60	4IAQ [136]	Class Aα	5-HT1B	Dihydroergotamine	Agonist
61	4IAR [136]	Class Aα	5-HT1B	Ergotamine	Agonist
62	4IB4 [137]	Class Aα	5-HT2B	Ergotamine	Agonist
63	4NC3 [138]	Class Aα	5-HT2B	Ergotamine	Agonist
64	5TVN [139]	Class Aα	5-HT2B	(8alpha)-N,N-diethyl-6-methyl-9,10-didehydroergoline- 8-carboxamide	Agonist
65	5CXV [140]	Class Aα	M1R	Tiotropium	Antagonist
66	3UON [141]	Class Aα	M2R	3-quinuclidinyl-benzilate	Antagonist
67	4MQS [142]	Class Aα	M2R	Iperoxo	Agonist
68	4MQT [142]	Class Aα	M2R	Iperoxo/LY2119620	Agonist/Allosteric modulator
69	4DAJ [143]	Class Aα	M3R	Tiotropium	Antagonist
70	4U14 [144]	Class Aα	M3R	Tiotropium	Antagonist
71	4U15 [144]	Class Aα	M3R	Tiotropium	Antagonist
72	4U16 [144]	Class Aα	M3R	N-methyl scopolamine	Antagonist
73	5DSG [140]	Class Aα	M4R	Tiotropium	Antagonist
74	5UEN [145]	Class Aα	A1AR	DU172	Antagonist
75	2YDO [147]	Class Aα	A2AR	Adensoine	Agonist
76	2YDV [147]	Class Aα	A2AR	NECA	Agonist
77	3EML [37]	Class Aα	A2AR	ZM241385	Antagonist
78	3PWH [146]	Class Aα	A2AR	ZM241385	Antagonist
79	3QAK [68]	Class Aα	A2AR	UK-432097	Agonist
80	3REY [146]	Class Aα	A2AR	XAC	Antagonist
81	3RFM [146]	Class Aα	A2AR	Caffeine	Antagonist
82	3UZA [149]	Class Aα	A2AR	6-(2,6-Dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-3-amine	Antagonist
83	3UZC [149]	Class Aα	A2AR	4-(3-amino-5-phenyl-1,2,4-triazin-6-yl)-2-chlorophenol	Antagonist
84	3VG9 [148]	Class Aα	A2AR	ZM241385	Antagonist
85	3VGA [148]	Class Aα	A2AR	ZM241385	Antagonist
86	4EIY [66]	Class Aα	A2AR	ZM241385	Antagonist
87	4UG2 [150]	Class Aα	A2AR	CGS21680	Agonist
88	4UHR [150]	Class Aα	A2AR	CGS21680	Agonist
89	5G53 [153]	Class Aα	A2AR	DB03719	Agonist
90	5IU4 [151]	Class Aα	A2AR	ZM241385	Antagonist
91	5IU7 [151]	Class Aα	A2AR	Compound 12c	Antagonist
92	5IU8 [151]	Class Aα	A2AR	Compound 12f	Antagonist
93	5IUA [151]	Class Aα	A2AR	Compound 12b	Antagonist
94	5IUB [151]	Class Aα	A2AR	Compound 12x	Antagonist
95	5K2A [152]	Class Aα	A2AR	ZM241385	Antagonist
96	5K2B [152]	Class Aα	A2AR	ZM241385	Antagonist
97	5K2C [152]	Class Aα	A2AR	ZM241385	Antagonist
98	5K2D [152]	Class Aα	A2AR	ZM241385	Antagonist
99	5UIG [154]	Class Aα	A2AR	5-amino-N-[(2-methoxyphenyl)methyl]-2-(3- methylphenyl)-2H-1,2,3-triazole-4-carboximidamide	Angatonist
100	3V2W [155]	Class Aα	S1P1	ML056	Antagonist
101	3V2Y [155]	Class Aα	S1P1	ML056	Antagonist
102	4Z34 [156]	Class Aα	LPA1	ONO9780307	Antagonist
103	4Z35 [156]	Class Aα	LPA1	ONO-9910539	Antagonist
104	4Z36 [156]	Class Aα	LPA1	ONO-3080573	Antagonist
105	5TGZ [157]	Class Aα	CB1	AM6538	Antagonist
106	5U09 [158]	Class Aα	CB1	Taranabant	Antagonist
107	3ZEV [160]	Class Aβ	NTSR1	Neurotensin	Agonist
108	4BUO [160]	Class Aβ	NTSR1	Neurotensin	Agonist
109	4BV0 [160]	Class Aβ	NTSR1	Neurotensin	Agonist
110	4BWB [160]	Class Aβ	NTSR1	Neurotensin	Agonist
111	4GRV [159]	Class Aβ	NTSR1	Neurotensin	Agonist
112	4XEE [161]	Class Aβ	NTSR1	Neurotensin	Agonist
113	4XES [161]	Class Aβ	NTSR1	Neurotensin	Agonist
114	5T04 [162]	Class Aβ	NTSR1	Neurotensin	Agonist
115	4ZJ8 [41]	Class Aβ	OX1	Suvorexant	Dual Antagonist
116	4ZJC [41]	Class Aβ	OX1	SB-674042	Antagonist
117	4S0V [163]	Class Aβ	OX2	Suvorexant	Dual Antagonist
118	5GLH [165]	Class Aβ	ET-B	Endothelin-1	Agonist
119	5T1A [167]	Class Aγ	CCR2	BMS-681/CCR2-RA-[R]	Orthosteric/allosteric antagonist
120	3ODU [168]	Class Aγ	CXCR4	IT1t	Antagonist
121	3OE0 [168]	Class Aγ	CXCR4	CVX15	Antagonist
122	3OE6 [168]	Class Aγ	CXCR4	IT1t	Antagonist
123	3OE8 [168]	Class Aγ	CXCR4	IT1t	Antagonist
124	3OE9 [168]	Class Aγ	CXCR4	IT1t	Antagonist
125	4MBS [170]	Class Aγ	CCR5	Maraviroc	Antagonist
126	5LWE [171]	Class Aγ	CCR9	Vercirnon	Antagonist
127	4EA3 [39]	Class Aγ	NOP	Banyu Compound-24 (C-24)	Antagonist
128	5DHG [172]	Class Aγ	NOP	Compound-35 (C-35)	Antagonist
129	5DHH [172]	Class Aγ	NOP	SB-612111	Antagonist
130	4DJH [173]	Class Aγ	κ-OR	JDTic	Antagonist
131	4DKL [174]	Class Aγ	μ-OR	Beta-FNA	Antagonist
132	5C1M [175]	Class Aγ	μ-OR	BU72	Agonist
133	4EJ4 [176]	Class Aγ	δ-OR	Naltrindole	Antagonist
134	4N6H [70]	Class Aγ	δ-OR	Naltrindole	Antagonist
135	4RWA [177]	Class Aγ	δ-OR	Bifunctional Peptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2)	Antagonist
136	4RWD [177]	Class Aγ	δ-OR	Bifunctional Peptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2)	Antagonist
137	4YAY [178]	Class Aγ	AT1R	ZD7155	Antagonist
138	4ZUD [179]	Class Aγ	AT1R	Olmesartan	Inverse Agonist
139	5UNF [180]	Class Aγ	AT2R	N-benzyl-N-(2-ethyl-4-oxo-3-{[2′-(2H-tetrazol- 5-yl)[1,1′-biphenyl]-4-yl]methyl}-3,4-dihydroquinazolin- 6-yl)thiophene-2-carboxamide
140	5UNG [180]	Class Aγ	AT2R	N-benzyl-N-(2-ethyl-4-oxo-3-{[2′-(2H-tetrazol- 5-yl)[1,1′-biphenyl]-4-yl]methyl}-3,4-dihydroquinazolin- 6-yl)thiophene-2-carboxamide
141	5UNH [180]	Class Aγ	AT2R	N-[(furan-2-yl)methyl]-N-(4-oxo-2-propyl- 3-{[2′-(2H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl]methyl}-3,4-dihydroquinazolin-6-yl)benzamide
142	4XNW [182]	Class Aδ	P2Y1	MRS2500	Antagonist
143	4XNV [182]	Class Aδ	P2Y1	BPTU	Antagonist
144	4NTJ [42]	Class Aδ	P2Y12	AZD1283	Antagonist
145	4PXZ [183]	Class Aδ	P2Y12	2MeSADP	Agonist
146	4PY0 [183]	Class Aδ	P2Y12	2MeSADP	Agonist
147	3VW7 [71]	Class Aδ	PAR1	Vorapaxar	Antagonist
148	5EE7 [78]	Class B	GCGR	MK-0893	Antagonist
149	4K5Y [186]	Class B	CRF1R	CP-376395	Antagonist
150	4Z9G [187]	Class B	CRF1R	CP-376395	Antagonist
151	4OR2 [188]	Class C	mGlu1	FITM	Negative Allosteric Modulator
152	4OO9 [38]	Class C	mGlu5	Mavoglurant	Negative Allosteric Modulator
153	5CGC [189]	Class C	mGlu5	HTL14242	Antagonist
154	5CGD [189]	Class C	mGlu5	HTL14242	Antagonist
155	4JKV [40]	Class F	SMO	LY2940680	Antagonist
156	4N4W [190]	Class F	SMO	SANT-1	Antagonist
157	4O9R [131]	Class F	SMO	Cyclopamine	Antagonist
158	4QIM [190]	Class F	SMO	Anta XV	Antagonist
159	4QIN [190]	Class F	SMO	SAG1.5	Agonist
160	5L7D [93]	Class F	SMO	Cholesterol
161	5L7I [93]	Class F	SMO	Vismodegib	Antagonist

Phylogenetic classification/structure

Prior to the availability of three-dimensional structural information, GPCR classification methods initially utilized primary amino acid sequences to phylogenetically categorize receptors. This approach ultimately laid the foundation for the most-commonly used GPCR classification system. The sequences of seven hydrophobic regions (represented as cylinders in figure 1A) were used to design a fingerprinting method to identify sequences not previously categorized as rhodopsin-like receptors [47]. In the method, an individual conserved hydrophobic region served as a single “feature.” Multiple features grouped together comprised a signature “fingerprint” within a sequence. A feature, when used by a scanning algorithm to search a database, is then referred to as a “discriminator” and fingerprints are referred to as “composite discriminators. The search results using the discriminators generate an output hit list for each hydrophobic region. A hit list correlation was used to differentiate between true members, where all features of the fingerprints matched, and noise, where zero, one or two random matches occur. A second database was built from the sequences resulting from the previous search and scans were repeated with the new discriminators. This process was repeated until the composite discriminator continuously distinguished between true members and noise. As a result, 240 sequences were identified as members of the superfamily. Sequences for pheromone receptors did not match any of the discriminators and cAMP receptors exhibited only two matches, thus falling within the noise [47]. These sequences were previously identified as GPCR [48] suggesting they were members of distantly related families [47]. The hit-list fingerprint correlation was later expanded to distinguish partial matches. Upon this expansion, sequences belonging the GPCR superfamily increased from 240 to 393. In addition, the pheromone, cAMP, and secretin-like families were established as rhodopsin-like receptors [49].

The rhodopsin-like family rapidly became overly complex as more diverse receptors were discovered, leading to the establishment of the GPCR superfamily and formation of the ‘class’ system. This A-F system includes GPCR found in both vertebrates and invertebrates [3]. In 2001, the first draft of the human genome became available [1, 2] and allowed further GPCR to be classified using the most prevalent classification system with most of the human GPCR categorized into five families shown in italics: Glutamate (class C), Rhodopsin (class A), Adhesion (class B), Frizzled/Taste2 (class F), and Secretin (class B); also recognized as GRAFS [3].

Rhodopsin (Class A)

The Rhodopsin (class A) family, the largest of the GPCR classes [3], is divided into α, β, γ, and δ subgroups [5]. The α-subgroup is the largest group in class A and is comprised of the prostaglandin, amine, opsin, melatonin, and MECA receptor clusters [3]. Generally, ligands bind to these receptors within a pocket inside the transmembrane cavity involving residues contained in TM3, TM5, and TM6 [50–53]. An example of this target binding domain can be found in the ADRB₂ structure in complex with carazolol (PDB: 2RH1) [26]. Biogenic monoamine receptors, such as the adrenergic; cannabinoid; muscarinic; serotonin; dopamine; and histamine receptors, are important drug targets for cardiovascular drugs, antipsychotics, and anti-histamines [54, 55]. Novel drugs that lack receptor specificity for particular amine receptors pose a risk for cardiovascular side effects due to off-target interactions with adrenergic receptors that show expression in many tissues [56]. Since the α-subgroup receptors outside the amine receptor subset are divergent from the amine receptors, side effects of novel aminergic drugs are most likely to occur only through the amine subset [5].

The β-subgroup of the Rhodopsin (class A) family has no branching subgroups and includes the hypocretin receptor, neuropeptide FF receptor, tachykinin receptor, cholecystokinin receptor, neuropeptide Y receptor, endothelin-related peptide receptor, gastrin-releasing peptide receptor, neuromedin receptor, thyrotropin releasing hormone receptor, ghrelin receptor, arginine vasopressin/oxytocin receptor, gonadotropin-releasing hormone receptor, and orphan receptors. Orphan receptors have been established as GPCR based on DNA sequence but have unidentified endogenous ligands [57]. Members of the β-subgroup mainly bind peptides with a high specificity-binding profile and are pursued as drug targets for treatments including pulmonary arterial hypertension [58] and hormone-related cancer [59]. Agonist drug design for β-subgroup members has been a challenge since it is difficult to design novel ligands that are flexible enough to mimic the magnitude of interaction sites engaged by endogenous peptide agonists [3].

The γ-subgroup of Rhodopsin (class A) contains the SOG, MCH, and chemokine receptor clusters, which bind both peptide and small ligand-like compounds. The SOG receptor cluster members include GPR54, the somatostatin receptors (SSTRs), and the opioid receptors [3]. The opioid receptors are important drug targets for the treatment of pain, cough, and alcoholism [54]. The MCH cluster includes receptors that branch off the SOG cluster. These receptors bind melanin-concentrating hormone (MCH), whereas the chemokine receptor cluster contains the chemokine receptors, the angiotensin/bradykinin-related receptors, and additional orphan receptors. Most ligands that interact with these receptors are peptides such as the chemokines and angiotensin. The chemokine receptors are drug targets due to their roles in acute and chronic inflammation [3] and as co-receptors engaged by some HIV strains [60]. While there are chemokine receptor-targeting drugs in clinical stages, maraviroc, an antagonist for CCR5, is the only currently FDA-approved drug on the market [61] targeting this class.

The δ-subgroup of Rhodopsin (class A) includes four main groups, the MAS-related receptor, glycoprotein receptor, purine receptor, and the olfactory receptor clusters. The MAS-receptor cluster includes the MAS1 oncogene receptor and the MAS-related receptors. The glycoprotein receptor cluster contains the classic glycoprotein hormone receptors and the leucine-rich-repeat-containing G protein-coupled receptors, whereas the purine receptor cluster is the largest in the δ-subgroup made up of the formyl peptide receptors, the nucleotide receptors, and a number of orphan receptors [3].

Extracellular region

The extracellular region of GPCR contains the N-terminus and three loops (ECL1-3) that shape the opening to the ligand-binding pocket. The ligand-binding region is found within the extracellular region of the 7TM bundle. EL2 (figure 5B) is known to vary in length between GPCR classes resulting in distinct conformations, while EL1 (figure 5A) and EL3 (figure 5C) are short and often have disordered structures [62]. A highly conserved disulfide bond between EL2 and C3.25 (see description of the Ballesteros-Weinstein numbering system in the 7TM region section) has been observed in a majority of crystallized GPCR structures. This covalent modification limits movement and stabilizes the conformation of EL2 [62, 63]. Compared to other classes, class A receptors have a shorter N-terminus [63].

Figure 5.

Superpositions of 31 class A (fuchsia), 2 class B (blue), 2 class C (yellow), and 1 class F (green) extracellular loops that are connected to the first two membrane-embedded helical turns are shown in ribbon structures where structures chosen did not have an N-terminal fusion partner. (A) EL1 and (C) EL3 are short in classes A through C and form mainly disordered structures that are mostly conserved among all classes. (B) EL2 is the longest of the EL, and varies in length and conformation in different GPCR. Class A GPCR: Rho (1F88), ADRB₁ (2VT4), ADRB₂ (2R4R), H₁R (3RZE), D₃R (3PBL), 5-HT_1B (4IAQ), 5-HT_2B (4IB4), M₁R (5CXV), M₂R (3UON), M₃R (4DAJ), M₄R (5DSG), A_2AR (3EML), S1P₁ (3V2W), LPA₁ (4Z32), CB₁ (5TGZ), NTSR₁ (3ZEV), OX₁ (4ZJ8), OX₂ (4S0V), TACR₁ (2KSP), CXCR1 (2LNL), CXCR4 (3ODU), CCR5 (4MBS), CCR9 (5LWE), κ-OR (4DJH), μ-OR (4DKL), δ-OR (4EJ4), HHV-5 US28 (4XT1), P2Y₁ (4XNW), P2Y₁₂ (4NTJ), PAR1 (3VW7), FFAR₁ (4PHU). Class B GPCR: GCGR (5EE7), CRF₁R (4K5Y). Class C GPCR: mGlu₁ (4OR2), mGlu₅ (4OO9). Class F GPCR: SMO (4QIN).

7TM region

Despite differences in primary structure, a majority of the proteins in class A/Rhodopsin share conserved residues found within the 7TM domain [5]. The Ballesteros-Weinstein numbering system is often used to assign numbers to common residues that are conserved in both sequence and structure-based alignments of different receptors [64]. This indexing system consists of two numbers separated by a period. The first value represents the TM helix in which the residue is found and has values ranging from 1 to 7. The second number denotes the residue position relative to the most conserved residue within that TM segment, which is defined as position 50. The value decreases as you move through the amino acid sequence toward the N-terminus and increases as you move though the amino acid sequence toward the C-terminus. Class A/Rhodopsin receptors have highly conserved residues in each TM segment: N1.50, D2.50, R3.50, W4.50, P5.50, P6.50, and P7.50 [64]. In addition to these residues, class A/Rhodopsin contains two fingerprint regions: the D/ERY motif at positions 3.49–3.51 and the NPXXY motif at positions 7.49–7.53 [3]. In 2000, x-ray crystallography studies on rhodopsin (PDB: 1F88) confirmed that conserved residues formed interhelical networks important for stabilization and activation [25] as had previously been suggested [65]. Seven years later, two structures of ADRB₂ (PDB: 2RH1, 2R4R/2R4S) were determined and revealed TM structural similarity to rhodopsin [26, 27]. The basic canonical architecture of this region has now been observed in numerous examples of crystallized GPCR as is shown in figure 4. It is interesting to note that an allosteric sodium ion binding pocket involving two conserved residues D2.50 and S3.39 was identified in the 1.8Å crystal structure of A_2A (PDB: 4EIY) [66, 67]. This central Na⁺ pocket was formed by D2.50, S3.39, and three water molecules. Liu, et al., compared their inactive A_2A structure to an active A_2A (PDB 3QAK [68]) structure and found that the size of the central pocket in the active form could only support three water molecules without sufficient room for Na⁺ coordination. This suggests that Na⁺ may stabilize the inactive conformation of A_2A [66]. After the discovery of the Na⁺ pocket in A_2A, the same characteristic was seen in other inactive GPCR crystal structures. These other structures include representatives of the α, γ, and δ subgroups of class A [ADRB1 (PDB: 4BVN [69]), delta-opioid (δ-OR; PDB: 4N6H [70]), and protease-activated receptor 1 (PAR1; PDB: 3VW7 [71])], suggesting the sodium ion binding pocket is a common feature in class A GPCR.

Conformational changes in the 7TM bundle are required for signal transduction across the cell membrane following ligand binding. Experimentally determined crystal structures of ADRB₂ reflect a variety of activation states. In these structures, TM5, TM6, and TM7 have a critical function in GPCR activation due to clear differences in helical arrangement. The active state of ADRB₂ (PDB: 3P0G) exhibits altered conformations of TM5 and TM7 and a prominent outward shift of TM6 [18] in contrast to the inactive state (PDB: 2RH1) [26].

The 7TM region forms distinctive ligand-binding pockets for different receptors where varying size, shape, and electrostatics provide receptor-ligand selectivity. Aminergic and nucleotide receptors have small binding pockets buried inside the 7TM bundle while peptide receptors have large, more accessible binding pockets near the extracellular surface [62]. These diverse ligand binding pocket profiles are demonstrated with 29 class A GPCR in figure 6A.

Figure 6.

Superpositions of GPCR represented as thin ribbon structures with ligands as space-filling atoms colored accordingly by antagonists as green, agonists as fuchsia, and negative allosteric modulators as orange. (A) Structures of 29 individual class A GPCR. (B) Structures of 3 class B GPCR. (C) Structures of 4 class C GPCR. (D) Structures of 7 class F GPCR. Class A GPCR: Rho (1F88), ADRB₁ (2VT4), ADRB₂ (2RH1), H₁R (3RZE), D₃R (3PBL), 5-HT_1B (4IAQ), 5-HT_2B (4IB4), M₁R (5CXV), M₂R (3UON), M₃R (4DAJ), M₄R (5DSG), A_2AR (2YDO), S1P₁ (3V2W), LPA₁ (4Z34), CB₁ (5TGZ), NTSR₁ (3ZEV), OX₁ (4ZJ8), OX₂ (4S0V), CCR4 (3ODU), CCR5 (4MBS), CCR9 (5LWE), NOP (4EA3), κ-OR (4DJH), μ-OR (4DKL), δ-OR (4EJ4), AT₁R (4YAY), P2Y₁ (4XNW), P2Y₁₂ (4NTJ), PAR1 (3VW7). Class B GPCR: GCGR (5EE7), CRF₁R (4K5Y, 4Z9G). Class C GPCR: mGlu₁ (4OR2, 5CGC, 5CGD), mGlu₅ (4OO9) Class F GPCR: SMO (4JKV, 4N4W, 4OR9, 4QIM, 4QIN, 5L7D, 5L7I).

Intracellular region

The intracellular region of GPCR includes the C-terminus and three loops (ICL1-3) that interact with G proteins, β-arrestins, and other downstream effectors [4, 10, 62]. GPCR crystal structures have shown structural conservation in the short IL1 chain, though high levels of variability have been observed within IL2 and IL3 suggesting dynamic and/or unstable conformations. Differences have been observed in IL2 in both the D₃R and ADRB receptor structures. In a crystal structure of D₃R (PDB: 3PBL) [6], where there were two copies of the protein from the same unit cell, IL2 had 2.5 turns of an α-helical conformation for chain A in contrast to a disordered loop lacking electron density for chain B (figure 7A). ADRB₁ [72] and ADRB₂ [26], which have an overall percent identity of 80% and nearly identical IL2 sequences, have displayed an α-helical conformation in ADRB₁ and unstructured conformation in ADRB₂ (figure 7B; PDB: ADRB₁ – 2VT4, ADRB₂ – 2RH1). The three dimensional structure of IL2 may be dependent upon the functional state of the protein, as well as interactions with intracellular partners. IL3 exhibits the greatest length variability amongst IL, ranging from five to hundreds of residues and has been implicated in G protein selectivity. IL3 251664384251663360 has been observed to form a disordered conformation or, more often, has been replaced by a fusion partner for increased conformational homogeneity to enable crystal formation in several solved GPCR structures [10]. There are crystallographic structures of rhodopsin (PDB: 3CAP) [73], ADRB₁ (PDB: 2YCW, 2YCX, 2YCY, 2YCZ) [74], and δ-opioid receptor (PDB: 4N6H) [70] where IL3 adopts an α-helical conformation resulting in extended TM5 and TM6 helices [10, 62]. The IL region undergoes considerable conformational changes required for G protein interaction and the consequent initiation of the signal transduction cascade.

Figure 7.

(A) Superposition of D₃R crystal structures (PBD: 3PBL) showing a 2.5 helical turn for chain A (purple) and no electron density for chain B (green) for IL2. The T4 lysozyme fusion partner replacing IL3 is shown in red for both chains. (B) Superposition of crystal structures comparing ADRB₁ (PDB: 2VT4 chain B; blue) and ADRB₂ (PDB: 2RH1 chain A; yellow and T4 lysozyme in red) where IL2 exhibits an ordered structure in ADRB₁ and a disordered structure in ADRB₂.

Secretin and Adhesion (Class B)

Class B, the second largest class within the Rhodopsin family, is comprised of the Secretin and Adhesion families. Secretin and Adhesion receptors have been classified together due to sequence similarities between their 7TM regions, although they have distinctions elsewhere that establish them as separate families. The Secretin family has an extracellular hormone-binding domain that interacts with peptide hormones [5]. The members of this family include the calcitonin/calcitonin-like receptors, the corticotropin-releasing hormone receptors, the glucagon receptor, the gastric inhibitory polypeptide receptor, the glucagon-like peptide receptors, the growth-hormone-releasing hormone receptor, the adenylyl cyclase activating polypeptide hormone receptor, the parathyroid hormone receptors, the secretin receptor, the vasoactive intestinal peptide receptors, and additional orphan receptors [3, 5]. The Secretin family includes potential targets for drug development due to their involvement in central homeostatic functions. Members of this family have been connected to appetite regulation and type-2 diabetes [5].

The Adhesion family includes the epidermal growth factor receptors and lectomedin receptors, though a majority of the members are currently classified as orphan receptors [5]. This GPCR family exhibits a highly variable number of amino acids at the N-terminal region, ranging from 200–2800 in length, making this family phylogenetically and structurally different from the rest of the class B GPCR. The “Adhesion” family name is related to the N-terminal region that contains sequence motifs, such as the GPCR proteolysis (GPS) motif, which serve as intracellular autocatalytic processing sites that participate in cellular adhesion [3, 5]. Adhesion family receptors bind extracellular matrix molecules rather than peptide hormones [5]. These receptors are believed to be involved in cell proliferation/migration, as well as immune system function via the mediation of leukocyte and neutrophil interactions. Adhesion receptors that contain long N-termini have become targets of monoclonal antibodies used as drugs candidates [5]. Numerous receptors from this family are localized in the central nervous system (CNS) [75] though their functional role in the CNS is not fully understood [5].

Structure

Within class B, sequence alignments show that Adhesion and Secretin families contain structural differences at the N-terminal region. Adhesion receptors contain distinctive O- and N-glycosylation sites, as well as EGF and GPS motifs [76]. Secretin family members have a 60–80 amino acid N-terminal domain [3] that include a hormone-binding (HRM) domain that is believed to have a key role in binding peptide hormones [63]. The Ballesteros-Weinstein numbering system somewhat extends to class B/Adhesion/Secretin where residues E3.50 and W4.50 are conserved [77]. The binding pockets of class B GPCR are broader and deeper inside the 7TM bundle compared to class A [45], as illustrated in figure 6B, in order to accommodate endogenous peptide ligands. On the other hand, the crystal structure for GCGR (PDB: 5EE7) shows an allosteric binding site outside of the 7TM domain between TM6 and TM7 [78].

Glutamate (Class C)

The Glutamate (class C) family of receptors consists of the metabotropic glutamate receptors, GABA receptors, single calcium-sensing receptors, and sweet and umami taste receptors. The known endogenous ligands of this family are known to bind to the N-terminal region, but many allosteric ligands have been discovered to interact with TM3, TM5, TM6, and TM7 [79–81]. In addition, Ca²⁺ can bind to the extracellular region [82] and enhance the effects of glutamate in some glutamate receptors [83, 84]. Many of the Ca²⁺ interacting residues are conserved within the “Venus flytrap” region of the Glutamate (class C) receptors [82] and have potential significance in drug design targeting depression learning, and memory [85].

Structure

In class C/Glutamate receptors, the 280–580 amino acid N-terminus [3] forms a cavity surrounded by two lobes which close upon ligand binding through a process known as the “Venus flytrap” mechanism (VFTM) [5, 86]. This mechanism involves a ligand binding-induced conformational change that results in the formation of disulfide bonds between the N-terminus and 7TM domain [63, 87]. Receptors in this class lack the conserved residues defined by the Ballesteros-Weinstein numbering scheme seen extensively in Class A and to a lesser extent in Class B GPCR. Although the conserved ligand binding pocket of most class C receptors is located within the extracellular region, there are allosteric binding sites within the 7TM bundle [5] as seen in figure 6C. These allosteric binding sites may be a way to achieve receptor specificity using allosteric modulators.

Frizzled/Taste2 (Class F)

The Frizzled/Taste2 (class F) group of receptors includes frizzled and smoothened receptors (SMO) [3, 5]. The frizzled receptors are known to bind secreted glycoproteins [88] while the SMO receptor functions in a ligand-independent manner through the SMO and sonic hedgehog (SHH) complex [89]. Frizzled receptors are involved in cell fate, proliferation, and polarity through association with secreted glycoproteins at the cysteine-rich N-terminus [3, 5]. Although the cysteine-rich region is highly conserved in the frizzled receptor, there is evidence that there are additional binding sites located in the extracellular loops of the TM regions [90]. SMO receptors are structurally similar to frizzled receptors [5]. The cysteine-rich region found in the frizzled receptors, as well as the chemical properties of residues that bind secreted glycoproteins, are conserved in SMO [90, 91]. Class F GPCR, though not well understood, have been linked to cancer development and are potential targets for cancer therapy [5].

Structure

Based on sequence analysis, class F/Frizzled/Taste2 is the most highly conserved class within the GPCR superfamily [63]. These proteins have a distinctive N-terminus that spans 200–320 amino acids in length [3, 5], in addition to a variable linker region between the EL and TM domains [5]. While the Ballesteros-Weinstein numbering scheme does not apply to this class, these proteins still share the common structure of the 7TM hydrophobic core [92]. There is limited structural information about this class of receptors since SMO is the only class F GPCR to be crystallized to date. The binding pocket is observed to be narrow and elongated in the currently available crystal structures (figure 6D). In one SMO crystal structure (PDB: 5L7D), cholesterol is bound to the extracellular cysteine-rich domain (CRD) that is highly conserved in vertebrates. An oxysterol was observed to displace cholesterol and bind to the CRD groove leading to SMO activation and Hedgehog (Hh) signaling. Cholesterol has been proposed as an endogenous ligand that stabilizes the inactive SMO conformation [93].

Conclusions/further perspectives

Since the initial crystallization of rhodopsin in 2000, numerous technological advances have significantly impacted GPCR structure determination efforts, resulting in crystal structures of 42 individual receptors (to date). Currently > 180 structures of GPCR have been solved and made available through the Protein Data Bank (table 1). Of these, over 150 protein-ligand complexes are available (table 2), representing the entire spectrum of ligand functions from inverse agonist to agonist. These data sets give insights into structural features and ligand recognition within this diverse protein superfamily. Specifically, the majority of crystallized ligand complexes with GPCR exhibit a ligand binding pocket near the extracellular end of the TM helical bundle, as shown in figure 6. The TM helical bundle structure shows considerable structural similarity across the family (figure 4), while EL2 shows the greatest structural diversity in the vicinity of the ligand binding pocket (figure 5). Thus, differences in ligand selectivity between GPCR family members are driven both by sidechain differences in the TM helical bundle (rather than backbone conformational differences) as well as overall EL2 fold. GPCR have essential biological roles, and many have been confirmed to have value as therapeutic targets. Therefore solved, three-dimensional GPCR structures have tremendous potential to influence drug discovery. Structure-based drug discovery approaches can be applied directly to crystallized GPCR family members as therapeutic targets. Additionally, the crystallized GPCR structures exhibit close sequence identity to additional GPCR family members and can serve as templates for the development of reliable and predictive homology models. Validated models allow structure-based drug discovery approaches to be used against this even broader set of target GPCR members. Although efforts in GPCR crystal structure determination over the past two decades have been fruitful, vast amounts of work remain to characterize unrepresented family members that have lower sequence identities to currently crystallized GPCR family members. GPCR structural characterization will continue to be a rich research area in need of further advances and innovative approaches into the foreseeable future.

Abbreviations

A_1AR

Adenosine A_1A receptor

A_2AR

adenosine A_2A receptor

ADRB₁

adrenergic β1 receptor

ADRB₂

adrenergic β2 receptor

AT₁R

angiotensin II receptor type 1

AT₂R

angiotensin II receptor type 2

CB₁

cannabinoid receptor 1

CCR2

chemokine receptor 2

CXCR4

chemokine receptor 4

CCR5

chemokine receptor 5

CCR9

chemokine receptor 9

CRF₁R

corticotropin-releasing factor 1

δ-OR

δ-opioid receptor

D₃R

dopamine D3 Receptor

ET-B

endothelin receptor type-B

CXCR1

chemokine receptor 1

EL

extracellular loop

FFAR₁

free fatty acid receptor 1

GPCR

g protein-coupled receptors

GCGR

glucagon receptor

mGlu₁

glutamate receptor 1

mGlu₅

glutamate receptor 5

H₁R

histamine H1 receptor

κ-OR

κ-opioid receptor

LPA₁

lysophosphatidic acid receptor 1

μ-OR

μ-opioid receptor

M₁R

muscarinic receptor 1

M₂R

muscarinic receptor 2

M₃R

muscarinic receptor 3

M₄R

muscarinic receptor 4

NTSR₁

neurotensin receptor 1

NOP

nociceptin receptor

OX₁

orexin receptor 1

OX₂

orexin receptor 2

PAR1

protease-activated receptor 1

P2Y₁

purinergic P2 receptor 1

P2Y₁₂

purinergic P2 receptor 12

Rho

rhodopsin

5-HT_1B

serotonin 5HT1B

5-HT_2B

serotonin 5HT2B

SMO

smoothened receptor

S1P₁

sphingosine 1-phosphate receptor 1

TACR₁

tachykinin receptor 1

TM

transmembrane

IL

intracellular loop

HHV-5 US28

viral GPCR US28