G-protein–coupled receptors (GPCRs) form a remarkable modular system that allows transmission of a wide variety of signals over the cell membrane, between cells and over long distances in the body.
G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes. These cell surface receptors act like an inbox for messages in the form of light energy, peptides, lipids, sugars, and proteins. Such messages inform cells about the presence or absence of life-sustaining light or nutrients in their environment, or they convey information sent by other cells.
GPCRs play a role in an incredible array of functions in the human body, and increased understanding of these receptors has greatly affected modern medicine. In fact, researchers estimate that between one-third and one-half of all marketed drugs act by binding to GPCRs.
Background
Every human cell is surrounded by a plasma membrane, a phospholipid bilayer. The membrane makes it possible for the cell to maintain a specific mix of biochemically active species, while preventing unwanted entry of other substances from the outside environment. For proper function, the biochemical machinery inside a cell needs to be able to receive instructions from the outside.
Changes in hormone levels on the outside of the cell elicit adaptive changes in enzyme activity on the inside. Odour molecules affect cells in the olfactory epithelium and substances in the food influence chemical activities in taste bud cells, which in turn elicit electrical signals that transfer information to the brain.
Indeed, human cells are constantly communicating with each other and the surrounding environment, which requires a molecular framework and a mechanism for transmission of information across the plasma membrane.
Moreover, in the body, signal transmission may take place over long distances. To be able to respond promptly, the brain needs rapid information from our senses, for vision, smell, taste and more. Again, this requires a molecular mechanism for transmission of information over the plasma membrane.
The molecular framework consists of G-protein–coupled receptors (GPCRs). Those are proteins located in the plasma membrane. The name GPCR refers to a common mode of receptor signaling via GTP-binding proteins on the inside of the cell.
Because their polypeptide chain passes seven times through the plasma membrane, the GPCRs are also called seven-transmembrane (7TM) receptors. They mediate a wide range of physiological signals from the outside of the cell.
The signal can be a change in concentration of peptides, hormones, lipids, neurotransmitters, ions, odourants, tastants, etc., or an influx of photons to the eye. GPCRs convey these signals to the inside of the cell and elicit a series of reactions involving other proteins, nucleotides and metal ions, which eventually deliver a message and an appropriate cellular and physiological response.
Many physiological processes in mammals depend on 7TM receptors, which are also the targets for a large portion of all pharmaceutical drugs. About a thousand human genes code for 7TM receptors, and they are involved in sensing a wide range of extracellular stimuli. Examples include the adrenergic receptors, dopamine receptors, histamine receptors, the light receptor rhodopsin, and the many odor and taste receptors.
Discovery of GPCRs
Most of what we know today about the intriguing molecular properties of 7TM receptors has been discovered over the past 40 years. However, rhodopsin had been identified as a photosensitive pigment as early as the 1870s, and its covalent ligand retinal was reported in 1933 .
The history of ligand-activated receptors started more than a century ago, when it was noted that reactive cells have a ‘receptive substance’ on their surface.
Early experiments with tissue preparations measured the responses to stimulators (agonists) and inhibitors (antagonists). During the following half century (about 1920–1970), classical receptor theory was developed, based on the law of mass action and dose-response data.
Some of the signaling components inside the cell were described in molecular detail before the receptors were. These include the second messenger cyclic AMP (cAMP) and the enzyme adenylyl cyclase, cAMP-dependent protein kinase and heterotrimeric G-proteins.
The nature of the ligand-activated receptors remained controversial up to the 1970s. The existence of receptors as separate molecular entities was debated, and it was even speculated that receptors and the enzyme adenylyl cyclase were the same protein.
Structure of GPCRs
Members of the GPCR superfamily share the same basic architecture of 7TM α-helices, an extracellular amino-terminal segment and an intracellular carboxy-terminal tail.
These plasma membrane-bound receptors have evolved to recognize a diversity of extracellular physical and chemical signals, such as nucleotides, peptides, amines, Ca2+ and photons. On recognition of such signals, the GPCRs act as proximal event in signaling pathways that influence a wide variety of metabolic and differentiated functions.
An extensive analysis of about 200 GPCR sequences revealed that the total length of GPCRs can vary between 311 and ∼1,490 amino acid residues. The largest variations in length are found in the N and C termini with size up to 879 and 371 amino acid residues, respectively.
GPCRs are not only encoded by eukaryotic genes but also by viral genes. Human GPCRs genomic genes are predominantly intronless.
The 7TM α-helices are connected by three intracellular and three extracellular loops. The extracellular loops of the GPCR can be glycosylated and contain two highly conserved cysteine residues, which build disulfide bonds to stabilize the receptor structure. GPCRs contain extracellular N-terminus domains (ECL1, ECL2 and ECL3) of variable size, ranging from 154 residues (calcitonin receptor) to 36 residues (rhodopsin receptor).
This domain contains asparagines residues and motifs for N-glycosylation, which influences intracellular trafficking of receptors to the plasma membrane, and cysteine residues in ECL1 and ECL2 loops that can influence protein folding critical for trafficking of a function receptor to the cell surface.19 The N terminus of some GPCRs is involved in ligand binding, activation and downregulation.
The 7TM α-helices helices of GPCRs are arranged to form a tight, ring-shaped central core that is highly hydrophobic in nature. Similar to most TM proteins, the hydrophobic amino acid residues are presumably arranged to face the lipid bilayer, whereas the more hydrophilic amino acid residues face towards the core.
Furthermore, helix-helix interaction contributes to the functional tertiary structure of the GPCRs necessary for receptor folding and stability, ligand binding and ligand-induced conformational changes for G protein coupling. Thus, mutations in the TM domain can have an array of deleterious effects.
GPCRs contain intracellular carboxyl-terminal domains (ICL1, ICL2 and ICL3) which are involved in several aspects of GPCR signaling. This domain contains Ser and/or Tyr residues which serve as sites for G protein receptor kinase-mediated phosphorylation and receptor desensitization.
Some GPCRs contain a cysteine residue in the C-terminal domain, which can serve as a site for palmitoylation. This can create a fourth IL (intracellular loops) because of the ability of the palmitoylated cysteine to insert in the plasma membrane. Also, C terminus may be involved in the interactions with other proteins that mediate GPCR signaling, such as the calcyon, PDZ domain-containing proteins, and Homer/Vesl proteins.19 The GPCR vary not only in sequence, but also in length of amino acid and carboxyl-termini (especially the C3 loop). The serine residue at the carboxy terminus region of GPCRs gets phosphorylated by G protein-coupled receptor kinases (GRKs).
GRKs constitute of six mammalian Ser/Thr protein kinase that phosphorylate agonist-bound, or activated, GPCR as their primary substrates, GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization.20 While the X-ray crystal structures of several GRKs have been solved, but the mechanism of GRK interaction with GPCRs was not known. Recently, many proposed a mechanism whereby the N-terminus of GRK2 protein forms an intramolecular interaction that selectively enhances the catalytic activity of the kinase towards GPCR substrates.
Recently, lot reported the 3.2 angstrom (A°) crystal structure of the bovine GPCR (opsin) in its G-protein-interacting conformation. (Ops-GalphaCT peptide complex). Recently report the crystal structure of ligand-free native GPCR (opsin) from bovine retinal rod cells at 2.9 A° resolution. Compared to GPCR rhodopsin, opsin showed some structural changes in the conserved E(D)RY and NPxxY(x)5,6F regions and in TM5–TM7. At the cytoplasmic side, TM6 was found to be tilted outwards by 6–7 A°, whereas the helix structure of TM5 was more elongated and close to TM6. The authors have suggested that the opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.
Family of GPCRs
These are the largest class of receptors, with more than one thousand GPCRs identified so far. The GPCRs family is the third most abundant family in Caenorhabditis elegans, comprising 5% of its genome with approximately 1,100 members. The Drosophila genome has at least 160 GPCRs. Based on the now entirely known human genome, careful estimation suggests that about 3–4% of the human genes code for GPCRs, about 1,200–1,300 members of the GPCR superfamily in the human genome, many of which are known to homo- and heterodimerize. However, in the case of plants a single GPCR has been isolated from pea and maize, and computational analysis shows their presence in Arabidopsis, Populus and rice. The GPCRs superfamily is divided into 6 major families which share little sequence homology among each other and some functional similarity. The 6 families of GPCRs are as follows:
Family A (rhodopsin receptor family): Family A is commonly known as rhodopsin family. It is the largest family of GPCRs and includes receptors for odorants and small ligands. This family is further divided into three groups. Group 1 contains GPCRs for small ligands including rhodopsin and β-adrenergic receptors. The binding site is localized within the seven TMs. Group 2 contains receptors for peptides whose binding site includes the N-terminal, the extracellular loops and the superior parts of TMs. Group 3 contains GPCRs for glycoprotein hormones. It is characterized by a large extracellular domain and a binding site which is mostly extracellular but at least with contact with extracellular loops e1 and e3.
Family B (secretin receptor family): Family B is commonly known as secretin family. It recruits about 60 members and is characterized not only by the lack of the structural signature present in family A but also by the presence of a large N-terminal ectodomain. Family B GPCRs have a similar morphology to group A3 GPCRs, but they do not share any sequence homology. Their ligands include high molecular weight hormones such as glucagon, secretine, calcitonin, growth hormone-releasing hormone, corticotropin-releasing factor, VIP-PACAP and the Black widow spider toxin, α-latrotoxin.
Family C (metabotropic glutamate receptors family): Family C recruits about two dozens GPCRs such as metabotropic glutamate receptors (mGluR) and the Ca2+ sensing receptors. This family also includes GABA-B receptors, taste receptors, olfactory receptors and a group of putative pheromone receptors coupled to the G protein Go (termed VRs and Go-VN). Like family B, these receptors possess large ectodomains responsible for ligand binding.
Family D (fungus pheromone receptor family): Family D comprises pheromone receptors (VNs) associated with Gi.
Family E (cAMP receptor family): cAMP receptors (cAR) have only seen found in D. discoideum but its possible expression in vertebrate has not yet been reported.
Family F (frizzled/smoothened receptor family): Family F includes the ‘frizzled’ and the ‘smoothened’ (Smo) receptors involved in embryonic development and in particular in cell polarity and segmentation
GPCR look like
GPCRs bind a tremendous variety of signaling molecules, yet they share a common architecture that has been conserved over the course of evolution. Many present-day eukaryotes — including animals, plants, fungi, and protozoa — rely on these receptors to receive information from their environment. For example, simple eukaryotes such as yeast have GPCRs that sense glucose and mating factors. Not surprisingly, GPCRs are involved in considerably more functions in multicellular organisms. Humans alone have nearly 1,000 different GPCRs, and each one is highly specific to a particular signal.
GPCRs consist of a single polypeptide that is folded into a globular shape and embedded in a cell's plasma membrane. Seven segments of this molecule span the entire width of the membrane — explaining why GPCRs are sometimes called seven-transmembrane receptors — and the intervening portions loop both inside and outside the cell. The extracellular loops form part of the pockets at which signaling molecules bind to the GPCR.
GPCR mechanism
As their name implies, GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein.
G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Some G proteins, such as the signaling protein Ras, are small proteins with a single subunit. However, the G proteins that associate with GPCRs are heterotrimeric, meaning they have three different subunits: an alpha subunit, a beta subunit, and a gamma subunit. Two of these subunits — alpha and gamma — are attached to the plasma membrane by lipid anchors.
A G protein alpha subunit binds either GTP or GDP depending on whether the protein is active (GTP) or inactive (GDP). In the absence of a signal, GDP attaches to the alpha subunit, and the entire G protein-GDP complex binds to a nearby GPCR. This arrangement persists until a signaling molecule joins with the GPCR. At this point, a change in the conformation of the GPCR activates the G protein, and GTP physically replaces the GDP bound to the alpha subunit. As a result, the G protein subunits dissociate into two parts: the GTP-bound alpha subunit and a beta-gamma dimer. Both parts remain anchored to the plasma membrane, but they are no longer bound to the GPCR, so they can now diffuse laterally to interact with other membrane proteins. G proteins remain active as long as their alpha subunits are joined with GTP. However, when this GTP is hydrolyzed back to GDP, the subunits once again assume the form of an inactive heterotrimer, and the entire G protein reassociates with the now-inactive GPCR. In this way, G proteins work like a switch — turned on or off by signal-receptor interactions on the cell's surface.
Whenever a G protein is active, both its GTP-bound alpha subunit and its beta-gamma dimer can relay messages in the cell by interacting with other membrane proteins involved in signal transduction. Specific targets for activated G proteins include various enzymes that produce second messengers, as well as certain ion channels that allow ions to act as second messengers. Some G proteins stimulate the activity of these targets, whereas others are inhibitory. Vertebrate genomes contain multiple genes that encode the alpha, beta, and gamma subunits of G proteins. The many different subunits encoded by these genes combine in multiple ways to produce a diverse family of G proteins
Secondary Messenger GPCR
Activation of a single G protein can affect the production of hundreds or even thousands of second messenger molecules. (Recall that second messengers — such as cyclic AMP [cAMP], diacylglycerol [DAG], and inositol 1, 4, 5-triphosphate [IP3] — are small molecules that initiate and coordinate intracellular signaling pathways.) One especially common target of activated G proteins is adenylyl cyclase, a membrane-associated enzyme that, when activated by the GTP-bound alpha subunit, catalyzes synthesis of the second messenger cAMP from molecules of ATP. In humans, cAMP is involved in responses to sensory input, hormones, and nerve transmission, among others.
Phospholipase C is another common target of activated G proteins. This membrane-associated enzyme catalyzes the synthesis of not one, but two second messengers — DAG and IP3 — from the membrane lipid phosphatidylinositol. This particular pathway is critical to a wide variety of human bodily processes. For instance, thrombin receptors in platelets use this pathway to promote blood clotting.
Conclusion
GPCRs are a large family of cell surface receptors that respond to a variety of external signals. Binding of a signaling molecule to a GPCR results in G protein activation, which in turn triggers the production of any number of second messengers. Through this sequence of events, GPCRs help regulate an incredible range of bodily functions, from sensation to growth to hormone responses.
