Activating movements

Introduction

G-protein-coupled receptors (GPCRs) form a large group of eukaryotic membrane-embedded cell surface receptors that relay information from the extracellular environment to the inside of the cell, thereby activating a variety of signaling cascades (1,2). GPCRs respond to numerous extracellular ligands, that include hormones, neurotransmitters, ions, photons and odorants.  GPCRs, therefore, mediate most physiological responses, ranging from vision, taste and smell to regulation of the immune system and behavior (1,2). Additionally, GPCRs play an essential role in disease such as the growth and metastasis of tumors (3) and it is therefore not surprising that GPCRs have emerged as an attractive therapeutic target for many diseases. In fact, it has been estimated that human GPCRs bind to about 30% of the small molecule drugs on the market (4). The superfamily of GPCRs contains about 600-1000 members making it the largest family of membrane proteins. Consequently, GPCRs are prominent within eukaryotic genomes. For example, the human genome encodes for about 800 GPCRs (around 15% of all human membrane proteins) (5). Vertebrate GPCRs are divided into five subfamilies based on their sequence and structural similarity. These are: rhodopsin, secretin, glutamate, adhesion and frizzled/taste2 (5). The rhodopsin subfamily is by far the largest with in humans 701 members. The secretin and glutamate receptor group contain both 15 members, while the adhesion and frizzled/taste2 receptor families comprise 24 members each.

Despite their functional diversity, all GPCRs share a common fold (figure 1 adapted from 6) that comprises seven transmembrane helices (H1-H7) that are connected by extracellular (E1-E4) and cytoplasmic (C1-C4) loops of varied length (1). The N and C-terminus are located on the extracellular or cytoplasmic side of the plasma membrane, respectively. GPCR-mediated cell signaling can be viewed as a two-step process, namely: initial GPCR stimulation through ligand binding that is coupled to the subsequent activation of specific signal-transducing G proteins that modulate downstream effector proteins (1,2). Based on their efficacy, GPCR ligands can be grouped in different classes e.g., full agonists that are capable of maximum receptor stimulation, partial agonists that are unable to completely activate a receptor, antagonists that don’t have an effect on receptor stimulation but prevent the binding of other ligands, and reverse agonists that decrease the activity of a receptor below that of a unliganded one (1). Signal-transducing G proteins typically form a heterotrimeric complex made up of an α, β and γ subunit. In addition, the α subunit exhibits GTPase activity that alternates between a GTP bound state (active) and one with GDP bound (inactive) (6). Following agonist binding and receptor activation, the heterotrimeric G protein complex associates with the receptor, and thereby resulting in displacement of GDP from Gα by GTP. Next, the α subunit dissociates from Gβ γ and activates downstream effector proteins (6). This raises the fundamental question of how receptor activation by agonist binding is mechanistically coupled to the association of the heterotrimeric G protein complex. To answer it, detailed structural information of liganded and unliganded GPCRs, preferably in association with G proteins, is required.  It is therefore important to note that recently high-resolution structures of the human serotonin receptor bound to different hallucinogens were presented (7), providing detailed molecular insight into GPCR activation as well as association with G proteins. Here I will discuss these structures and their current functional understanding.

GPCR-mediated signal transduction

The vast majority of cellular responses to external stimuli such as light, hormones and neurotransmitters are mediated by GPCRs. The classical GPCR-dependent signaling cycle is shown in figure 2 (adapted from 9), which is initiated by binding of an agonist to its cognate receptor. This stabilizes the GPCR in the active conformation and involves an outward shift of transmembrane domain (TM) 6, enabling it to interact with heterotrimeric G proteins. These comprise an α, β and γ subunit. Both the α and γ subunits are peripherally associated with the membrane via their covalently attached lipids. Functionally, G proteins are characterized by their α subunit and the human proteome contains more than 21 different Gα subunits that are encoded by 16 genes. These Gα proteins are subdivided into four major families according to sequence and functional similarity, namely: Gα_s, Gα_i, Gα_q and Gα₁₂. Gα_s (stimulation) subunits are expressed in most cells, while a specialized variant is specifically expressed in olfactory neurons. Gα_i (inhibition) proteins form the largest and most diverse family of which the members are commonly found in most cells. Gα_q subunits are ubiquitously present in all cells, while the expression of specialized variants is restricted to the lung, kidney, liver and hematopoietic cells. Members of the Gα₁₂ family are expressed in most cells. Moreover, the human genome contains five genes encoding Gβ subunits and 12 genes encoding Gγ subunits.

Gα proteins display GTPase activity and function as a molecular switch, shuttling between an active (with GTP bound) state and an inactive (with GDP bound) state. Association of the heterotrimeric G protein with the GPCR triggers release of GDP from Gα and subsequent binding of GTP. Next, Gα dissociates from the β and γ subunit, which are tightly associated and can be regarded as one functional unit. It has been established that Gα and Gβγ target different downstream effector proteins. Gα is responsible for the stimulation of downstream effector proteins such as adenyl cyclase and phospholipase C, whereas Gβγ activate GIRK-type (G-protein-coupled inwardly rectifying) potassium channels. Cellular signaling is terminated by hydrolysis of GTP and rebinding of Gα to Gβγ, resulting in the inactive heterotrimer and thereby completing the GPCR-dependent signaling cycle. Interestingly, GPCRs can activate different signaling pathways, while ligands are able to stimulate signaling systems to a different extent. For example, opposite activities for different signaling pathways have been observed.                                 

Biological and structural features of GPCRs

Despite their key role in many physiological responses and their relative abundance in the eukaryotic proteome, all GPCRs share a common protein fold ranging between 200 to 1000 residues with seven transmembrane domains connected by extracellular and cytoplasmic loops (figure 1). In general, ligands bind to the extracellular loops and N-terminus, whereas the cytoplasmic part is involved in the binding of G-proteins or other subunits. GPCRs are, therefore, allosteric proteins – agonist binding at the extracellular binding site (the orthosteric site) promotes binding of a G-protein at the cytoplasmic side (2). It is commonly assumed that GPCRs are functional in a monomeric form, which was confirmed by functionally reconstituting purified µ-opioid receptor in vitro into high density lipoproteins (10). This showed that oligomerization is not required for ligand binding, while the monomeric receptor is sufficient for G-protein activation. However, several lines of biochemical evidence indicate that GPCRs from various families exist as dimers or higher order oligomers. The human proteome contains about 800 GPCRs of which the majority belongs to the subfamily of rhodopsin-like receptors (5). 

These represent the best characterized receptors and exhibit a high degree of sequence conservation. Figure 3 (adapted from 11) displays a general topological model of rhodopsin-like GPCRs with the most conserved residues in each TM in bold. In addition to the characteristic 7 TMs, rhodopsin-like receptors contain a C-terminal helical segment (H8) that is probably located parallel to the cytoplasmic face of the membrane and contains two palmitoylated cysteines that link it to the membrane. Within their TMs, rhodopsin-like GPCRs contain 25 conserved residues of which some are part of important motifs. These include the DERY motif in TM3, CWXP motif of TM6, and NPXXY motif of TM7 (11). These are critical for receptor activation. Moreover, GPCRs display a high degree of conformational flexibility, meaning that they display a continuum of conformations with comparable stabilities (2). Ligand binding stabilizes one particular conformation that is able to interact with G-proteins. This raises one crucial question: how does binding of an agonist triggers changes in interactions at the ligand binding pocket that result in conformational changes which are propagated from the extracellular ligand binding pocket to the cytoplasmic part of the protein involved in G-protein binding (1). Detailed structures of GPCRs in the inactive and active state have provided an answer to this question. These are discussed below in more detail.                                                                                                        

Structure of an inactive GPCR

Important molecular insight into the activation of GPCRs was obtained through structural analysis of rhodopsin. This GPCR is found in rod cells and is activated by light, thereby stimulating the signaling pathway that leads to vision (6). Rhodopsin is made up of the protein opsin and a light-absorbing pigment known as 11-cis-retinal, that functions as an inactivating ligand. This compound is a vitamin A derivative that is covalently linked to opsin. Following absorption of a photon, 11-cis-retinal is isomerized to all trans-retinal. This induces a conformational change of opsin, resulting in activation of the receptor. However, all trans-retinal is short lived because it is quickly hydrolyzed and therefore only transiently activates opsin. Rhodopsin is recycled via loading with newly synthesized 11-cis-retinal. The absorption of a single photon activates hundreds of G-proteins, enabling humans to detect a flash of only five photons. In 2000, the crystal structure of inactive bovine rhodopsin at 2.8 Å resolution was presented (12). This is shown in figure 4 in ribbon representation with conserved motifs in TM3, TM6 and TM7 as spheres (left and center panel). The structure of inactive rhodopsin clearly reveals the conserved fold of all GPCRs with seven TM helices followed by a short helical segment (H8) that is located parallel to the cytoplasmic membrane. Several of these TMs such as TM5, TM6 and TM7 contain conserved helix breaking proline residues. This results in the characteristic presence of kinks in these TMs. Within the inactive structure, TM3 and TM7 are linked by a salt bridge between Lys296 and Glu113 which represents the so-called 3-7 lock which is shown in the center panel (top view) of figure 4. Rhodopsin contains three extracellular loops (EI, EII and EIII) of varying length that connect the TMs and associate to form a compact structure. The extracellular N-terminal tail comprises five distorted strands of which some are folded into a typical β-sheet (center panel). The extracellular loops contain two N-linked oligosaccharides (Asn2 and Asn15) that are not involved in any interactions. Loop EI and EIII run along the surface of the receptor (left panel). In most rhodopsin-like receptors EII is stabilized by a disulfide bridge and it contains a part that is made up two β-sheets. These dip into the center of rhodopsin and the lowest one of these β-sheets is part of the retinal-binding site. This arrangement of EII ensures an extensive contact with the other extracellular regions and retinal. Retinal is shown in blue sticks and is sandwiched in between TM3, TM5, TM6 and TM7, which make up the ligand-binding site (right panel). The chromophore is covalently attached to the protein backbone via Lys296 (as green ball and stick) that forms a protonated Schiff base, which is stabilized by Glu113 (red ball and stick).

This ionic interaction connects TM3 and TM7 and is known as the 3-7 lock (center panel). Additional residues that are part of the retinal binding pocket and belong to TM3 include Gly114, Ala117, Thr118, Gly120 and Gly121 (orange ball and stick). Rhodopsin contains three cytoplasmic loops (CI, CII and CIII) that connect the TMs as well as a C-terminal tail (left panel). This part relatively long and well exposed into the cytoplasm. Based on its high B-factor, the C-terminal tail is flexible which also explains why it is not completely resolved. In contrast, CI and CII are much shorter and more rigid with CII adopting an L-shaped conformation. Additionally, the hydrophobic residues at the cytoplasmic termini of TM2, TM5, TM6 as well as CII form a large part of the G-protein binding site. CIII is also flexible and is localized near the C-terminal tail. Interestingly, CIII displays little sequence conservation amongst rhodopsin-like receptors, indicating that it may be important for functionality and specificity of G-protein activation. TM7 is connected to a small helical peptide (H8) that runs along the inner face of the cytoplasmic membrane and contains two palmitoylated cysteines that link it to the membrane. Moreover, it has been established that this helix directly interacts with G-proteins and contains a cluster of highly conserved residues, suggesting that H8 is functionally important for GPCRs.

Structure of an active GPCR

Figure 5 presents the structure of apoopsin in ribbon representation that was determined in 2008 at 2.9 Å resolution (13) in an active-like conformation. Overall, the architecture of the ligand-free receptor (left panel) is similar to that of rhodopsin with seven TMs that are connected by three cytoplasmic loops and three extracellular loops. The structure also includes the cytoplasmic helix (H8) downstream of TM7 that runs parellel to membrane as well as two glycan chains attached to Asn2 and Asn15 and palmitoylated Cys322, which anchors H8 to the membrane. As expected, the salt bridge between Lys296 and Glu113, that connects TM3 and TM7 (3-7 lock), is not present in the absence of 11-cis-retinal (right panel with Lys296 and Glu113 as magenta spheres), a stong inverse agonist. It was proposed that this interaction must be broken in order to activate the receptor and therefore the structure of the aporeceptor is in good agreement with an active-like conformation. 

A structural comparison between inactive rhodopsin and its apo variant is shown in figure 6, revealing only small changes for TM1-TM4, while larger conformational changes are observed for TM5-TM7, especially in the cytoplasmic ends of these TMs (center panel with for rhodopsin (PDB 1F88) TM3 in grey, TM5 in yellow, TM6 in purple and TM7 in pink. In opsin (PDB 3CAP), TM3 is in orange, TM5 in ruby, TM6 in grey and TM7 in green). The most prominent changes include an outward shift of TM6 and an elongation and a sideward rotation of TM5. Other crucial conformational changes occur in the conserved sequence motifs (right panel with respective residues of inactive rhodopsin (PDB 1F88) as purple sticks or grey sticks for the apo receptor (PDB 3CAP)). Specifically, in the inactive receptor a salt bridge is observed between Arg135 of TM3 (of the DERY motif) and Glu247 of TM6 as well as Glu134 of TM3 and Thr251 of TM6. These interactions form the so-called ionic lock and stabilize TM6 within the transmembrane part of the receptor, thereby keeping rhodopsin in the inactive conformation. The ionic lock is broken in the aporeceptor which results in the rearrangement of the cytoplasmic ends of TM5 and TM6. Conformational changes of TM5 and TM6 are consistently observed in the structures of activated GPCRs which include a rotation of conserved residues (11). In opsin, for example, Trp265 of TM6 that is part of the CWXP motif is oriented outward towards TM5 along with Phe261. The joint reorientation of these residues results in the horizontal rotation of TM6.  While in rhodopsin these residues face inwards towards TM3 within the transmembrane bundle. The reorientation of Trp265 and Phe261 links the agonist binding site with the conformational change of TM5 and TM6 via a rearrangement of TM3, TM5 and TM6. TM7 contains the NPXXY motif that connects TM7 and H8 through an electrostatic interaction between Tyr306 and Phe313.Within opsin, this interaction does not occur because TM7 is oriented in such a way that Tyr306 points towards the transmembrane domains and thereby preventing TM6 moving back towards TM3 as observed in rhodopsin. 

Within inactive rhodopsin, the CWXP, NPXXY and DERY motifs are separated from a water-mediated hydrogen bond network between TM6 and TM7 that is essential for G-protein activation by a hydrophobic barrier made up of six residues (left panel of figure 7) in TM2 (Leu76, Leu79), TM3 (Leu128, Leu131 and TM6 (Met253, Met257) (11). The outward movement of TM6 during receptor stimulation opens the hydrophobic network (right panel of figure 7), which disrupts the hydrogen bond network. This, in turn, allows the reorientation of Tyr306 (of the NPXXY motif) and Tyr206 (of TM5) in between TM3 and TM6 to extent the hydrogen bond network towards the DERY motif. These have been proposed to act as toggle switch, controlling the transition of GPCRs between an on and off state during signal transduction. In sum, the outward movement of TM6 and the subsequent disruption of the hydrogen bond network between TM6 and TM7 results in an extended network that connects residues from the retinal binding site all the way up to cytoplasmic residues of the G-protein binding site (11).   

Based on the structural data discussed above, four molecular switches have been identified in (rhodopsin-like) GPCRs, namely: ionic lock switch, 3-7 lock switch, transmission switch (CWXP motif), that controls the horizontal rotation of TM6, and tyrosine toggle switch (NPXXY motif), which regulates the extension of the hydrogen bond network (11,14). The activation of GPCRs can, therefore, be viewed as a series of sequential steps in molecular switches located in conserved microdomains. Mechanistically, these switches are critical for receptor activation through relaying structural changes induced by agonist binding at the extracellular side towards cytoplasmic residues that control the activation and binding of G-proteins. 

Structure of hallucinogen-activated human serotonin receptor

Human HTR2A is a rhodopsin-like GPCR that is a member of the serotonin receptor family. It is widely expressed throughout the central nervous system especially in brain regions for learning and cognition. In fact, its signaling pathway has been implicated in the modulation of working memory and attention through controlling the release of neurotransmitters and hormones. HTR2A is also medically relevant because its dysfunction is associated with different psychiatric disorders, including depression, schizophrenia and drug addiction (15). Serotonin represents the physiological agonist of HTR2A, while it also activated by a number of psychedelics and hallucinogens such as LSD, psilocin and mescaline. Additionally, synthetic hallucinogens that are based on N-benzyl-2,5-dimethoxy-phenetylamine (25CN-NBOH) represent potent agonists. In order to understand how psychedelics and hallucinogens exert their actions, it is important to determine how they interact with HTR2A. Therefore, the structure of this GPCR bound to 25CN-NBOH and in complex with Gαβγ was determined at 3.3 Å by Cryo-EM (8). A ribbon representation of this structure is shown in the top left panel of figure 8 with HTR2A in green, Gαq in blue, Gβ in pink and Gγ in yellow. Within this complex, HTR2A is folded into the characteristic GPCR conformation with seven TMs that are connected by three extracellular and cytoplasmic loops of different length, respectively. To stabilize this structure, an engineered variant of Gαq was used (mini-Gαq) in which the first 35 residues were replaced by the corresponding sequence of another α subunit. The overall structure of Gαq is similar to that of other Gα proteins with a RAS-like GTPase domain, a typical α/β nucleotide-binding fold made up of α-helices and antiparallel β-sheets, as well as a helical domain (7). The β and γ subunits form a dimer that behaves functionally as a monomer. Gαq associates with Gβ through its N-terminal helix. The β subunit is folded into a β propeller with seven blades that are each made up of four antiparallel β-strands (7). Additionally, Gβ contains an N-terminal helix that interacts tightly with Gγ. The γ subunit is much smaller than Gβ and requires a tight interaction with the β subunit to fold. In fact, in the absence of Gβ the γ subunit is unstable and rapidly degraded. Within the Gβγ dimer, the γ subunit is wrapped around the outside of Gβ with its two α-helices in a coiled-coil interaction (7). A close-up of the interaction interface between HTR2A and the N-terminal helix of Gαq is provided in the top right panel of figure 8 with HTR2A residues as grey ball and sticks and Gαq residues as purple ball and sticks. HT2RA residues Asn107, Asp172, Asn317 and Asn384 are connected through hydrogen bonds with Glu242, Tyr243, Gln237 and Asn244 of Gαq. Furthermore, residues Ala321, Leu261, Ile177, Lys325 and Val324 of HT2RA form a hydrophobic core with Leu236, Leu240 and Leu245 of Gαq. The importance of these residues was analyzed by replacing them with alanine, showing that the receptor was inactivated by the mutation Gln237Ala in Gαq. This is a conserved residue in proteins belonging to the Gαq group and it may therefore be important for determining Gαq specificity. Additionally, Ile181 is highly conserved among GPCRs and its thought to be crucial for the binding of Gαq. It is therefore not surprising that its replacement with either alanine or glutamic acid abolished the activity of HTR2A to stimulate Gαq. 

Within the structure of HTR2A, the agonist, 25CN-NBOH, was well resolved (in orange ball and sticks in the bottom panel of figure 8 and its chemical structure in the center panel). It is located at the extracellular side of the receptor sandwiched in between the TMs. A close-up of the ligand-binding pocket is provided in the right bottom panel of figure 8 with 25CN-NBOH in orange ball and sticks and interacting residues in raspberry ball and sticks. A crucial interaction comprises a salt bridge between Asp155 and a positively charged nitrogen of 25CN-NBOH. It has been established that mutation of this residue abolishes ligand binding. Additional hydrophobic interactions involve Val156, Val235, Trp336, Phe339 and Phe340. Many of these residues are probably important for ligand binding and receptor activation. Moreover, Trp336 is associated with 25CN-NBOH through a π-π interaction, while a hydrophobic interaction is observed between Ser159 and the 2-OH group of the ligand. Likewise, a similar interaction occurs between 2-OH group of the ligand and Gly369. Replacement of these residues with alanine inhibits the potency of 25CN-NBOH. Interestingly, Gly238 is not well conserved among receptors that bind other biogenic amines. Instead, these GPCRs often contain a Ser or Thr at this position and replacement of Gly238 in HTR2A with Ser reduced the potency of 25CN-NBOH. This residue, therefore, represents one of the structural features that is essential for the selective activity of 25CN-NBOH.

Summarizing conclusion

GPCRs represent the largest family of eukaryotic membrane proteins with about 600-1000 members. In humans, GPCRs control almost every physiological aspect because they activate a variety of cellular signaling pathways in response to stimulation by extracellular ligands such as hormones, neurotransmitters, ions, photons and odorants. The human genome encodes for about 800 GPCRs (around 15% of all human membrane proteins) (5) that are divided into five subfamilies based on their sequence and structural similarity. These are: rhodopsin, secretin, glutamate, adhesion and frizzled/taste2 (5). The rhodopsin subfamily is by far the largest with in humans 701 members. Despite their functional variety, all GPCRs share a common protein fold with seven transmembrane domains connected by extracellular and cytoplasmic loops. The extracellular loops and N-terminus are involved in ligand binding, while the cytoplasmic part is involved in the binding of G-proteins or other subunits (1). Owing to these two binding sites, GPCRs are typical allosteric proteins. This because agonist binding at the extracellular binding site (the orthosteric site) promotes binding of a G-protein at the cytoplasmic side (2). Biochemical and structural studies of different GPCRs have established that receptor activation follows agonist binding and involves an outward rotation of TM5 and TM6 (1,2). However, the initiation and sequence of these molecular events as well as the residues involved are not well understood. A recent computational study proposed a common activation mechanism for human rhodopsin-like GPCRs (16). In detail, it was suggested that the pathway that links residues within the ligand binding site to the G-protein binding region involves four layers of residues (figure 11 adapted from 16). The first layer comprises residues that, in response to the binding of extracellular ligands, trigger an alteration of the transmission switch (CWXP motif) as well as a collapse of the sodium pocket that is present in inactive rhodopsin-like GPCRs. The changes within the CWXP motif initiate the rotation of the cytoplasmic ends of TM6, while the collapse of the sodium pocket initiates the movement of TM7 towards TM3. Alterations in layer 2 occur simultaneously with those in the first layer and concern changes in residues of the hydrophobic barrier (lock) in TM3 and TM6 that trigger its opening. 

This loosens the interactions between TM3 and TM6, thereby facilitating the outward movement of the cytoplasmic end of TM6. Additional changes of residues in TM7 drive its movement towards TM3. Layer 3 comprises changes in residues of the NPXXY motif that strengthen the interactions between TM3 and TM7 and further loosen the interactions between TM3 and TM6 as the latter moves outward. Layer 4 concerns structural changes in residues of the DERY motif and disruption of the ionic lock. These drive the outward movement of the cytoplasmic end of TM6 and rewire residues within TM3, TM5 and TM6 that serve as G-protein contacting positions, which renders the receptor competent for G-protein binding. Importantly, human GPCRs are involved in many diseases and represent therefore important drug targets. In fact, about one third of all marketed drugs are directed against human GPCRs. Therefore, an increased molecular understanding of GPCR activation is expected to aid in the design of novel small molecule drugs that selectively modulate receptor activity as well as improve the pharmacology of marketed therapeutics that target GPCR function (4).

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