Class B GPCRs
G protein coupled receptors (GPCRs) are the largest class of integral membrane proteins. GPCRs are divided into five families; the rhodopsin family (class A), the secretin family (class B), the glutamate family (class C), the frizzled/taste family (class F), and the adhesion family.[1] Roughly 5% of the human genome encodes g protein-coupled receptors, which are responsible for the transduction of endogenous signals and the instigation of cellular responses.[1] All GPCRs contain a similar seven α-helical transmembrane domain that once bound to its ligand, undergoes a conformational change and tranduces a signal to coupled, heterotrimeric G proteins. The initiation of intracellular signal pathways occur in response to stimuli such as light, Ca2+, amino acids, nucleotides, odorants, peptides, and other proteins, and accomplishes many interesting physiological roles. [1]
Class B GPCRs contain 15 distinct receptors for peptide hormones and generate their signal pathway through the activation of adenylate cyclase (AC) which increases the intracellular concentration of cAMP, inositol phosphate, and calcium levels. [2] These secondary messengers are essential elements of intracellular signal cascades for human diseases including type II diabetes mellitus, osteoporosis, obesity, cancer, neurological degeneration, cardiovascular diseases, headaches, and psychiatric disorders; making their regulation through drug targeting of particular interest as disease targets. [3] Structural approaches to the development of agonists and antagonists have however been hampered by the lack of accurate Class B TMD visualizations. Recent crystal structure images of corticoptropin-releasing factor receptor 1 (PDB: 4K5Y) and human glucagon receptor (PDB: 4L6R) were accomplished through x-ray crystallography. [4] [5]
Glucagon Receptor (GCGR)
The glucagon class B GPCR (GCGR) is involved in glucose homeostasis through the binding of the signal peptide glucagon. Glucagon is released from pancreatic α-cells when blood glucose levels fall after a period of fasting or several hours following intake of dietary carbohydrates.[6] Once the peptide hormone is released, it binds to GCGR, a 485 amino acid protein found in the liver, kidney, intestinal smooth muscle, brain, and adipose tissues. [7] Upon binding, signaling is initiated to heterotrimeric G-proteins containing Gαs. [8] GCGR can regulate additional signal pathways, including G-proteins of the Gαi family through the adoption of differing receptor conformations. [9]
Glucagon's main role is the regulation of blood glucose levels.[6] Glucagon lowers the concentration of fructose 2,6-bisphosphate which is an allosteric inhibitor of the gluconeogenic enzyme fructose 1,6-bisphosphotase and activates phosphofructose kinase 1, which increases glucose levels via glycolysis.[6] Glucagon is also a regulator of the production of cholesterol, which is an energetically intensive process. When energy resources are low, downregulation of cholesterol production begins with glucagon binding to GCGR, which stimulates the phosphorylation of HMG-CoA.[6] HMG-CoA is inactivated by phosphorylation and moderates cholesterol production to conserve energy.[6] Glucagon also takes part in fatty acid mobilization by affecting levels of adipose tissue in the organism. Activation of GCGR by glucagon initiates triacylglycerol breakdown and the phosphorylation of perilipin and lipases via cAMP signal pathways.[6] This allows the body to export fatty acids to the liver and other crucial tissues for energy use and makes more glucose available for use in brain functioning.[6]
Structure
The class B GPCRs, including GCGR, are different from other GPCRs in several ways. The first is that class B GPCRs contain a protrusion known as a 'stalk,' a three α-helical turn elongation of the N-terminus that protrudes past the extracellular (EC) membrane.[5] Structural integrity of this domain in GCGR is A135P mutations impact stalk stability by removing an important salt bridge between Glu133 - Lys136.[5] A second difference between class B and other GPCRs is that the extracellular loop 1 (ECL1) is 3-4 times longer than comparable loops in class A GPCRs, and also affects ligand binding affinity.[5] Most notably, class B GPCRs contain a which is solvent filled and accessible from the extracellular side.[3] This central splay is notably absent , and represents a tantalizing target for agonists/antagonists.[3]
Because of the difficulty in stabilizing and crystallizing Class B TMDs, very little is known about the conformational changes that transduce cell signals endogenously. GCGR is known to regulate additional signal pathways through the adoption of differing receptor conformations and to interact with receptor activity-modifying proteins (RAMPs) altering the signaling bias of the receptor.[9]
Glucagon Binding
Fig. 1: Snake Plot of GCGR TMD. Residues of particular importance in glucagon binding affinity are found in green, yellow, and black. Residues in red are the location of critical disulfide bonds, while blue residues were found to be highly conserved across all class B GPCRs.
[5]
Fig. 2: functioning as anchoring site for glucagon's n-terminal residues.
Fig. 3: Surface visualization of glucagon visualizing the three dimensional shape of the N-terminal tail that interacts with the binding site of GCGR central cavity.
The large, soluble N-terminal extracellular domains (ECD) of GCGR provide initial ligand selectivity with the deep, ligand pocket (Fig. 2) of the TMD providing secondary recognition.
[7] In a comprehensive mutagenesis and glucagon-binding study, a total of 129 mutations of GCGR were tested. 41 of these covering 28 different locations in the GCGR TMD were found to have at least a fourfold decrease in glucagon binding affinity
[5]. (see Fig. 1) It is the face of the central cavity that harbors the majority of the residues which play an important role in glucagon binding.
[5] The binding site was shown to be a
dynamic area traveling from the middle of the stalk region (Tyr 138) to deep within the 7TM core (Glu 362), encompassing positions along ECL1, ECL2 and ECL3 and helices I, II, III, V, VI and VII.
Mutagenesis and photo cross-linking studies determined essential, conserved residues in glucagon and have been in red.[5] Glucagon residues His 1, Gln 3, Phe 6, and Tyr 10 are critical to successful binding interaction with the GCGR while others are important for structural rigidity. The n-terminus of glucagon (Fig. 3) leads to a protuberance that fits into the deep, interior cavity of the GCGR 7TMD (Fig. 2) where four residues reside that play strong roles in ligand binding affinity. There is a to the entrance of the cavity, providing a firm anchor during peptide docking. (also see Fig. 2)
Clinical relevance
Figure 4: Three small molecule antagonists reported in 2007.
[10].
Because GCGR can interact with multiple types of G protein subfamilies, discovering small molecule inhibitors could lead to a wide range of focused therapies.[11] Blocking conformations that favor interaction with specific G proteins could allow the knockdown of targeted signal pathways. For example, GCGR is known to interact with inhibitory Gαi proteins that antagonize cAMP production.[11] The finding of an agonist for this pathway could lead to breakthroughs in the treatment of diabetes mellitus. Recently some fundamental work has been done with RAMPs which were shown to alter ligand preference in class B GPCRs.[12] Specifically, RAMP2 association has been shown to alter the pharmacology of all GCGR ligands (glucagon and oxyntomodulin). RAMP2 association altered cAMP production, indicating an effect on signaling bias and g protein coupling.[12]
Attempts to target the GCGR have proven relatively unsuccessful. Three small molecule modulators were reported with the hope of enhanced pharmaceutical regulation.[10] (Fig. 4) It is not clear if this work has resulted in additional pharmacological modalities, but any progress has been modest, at best. Some gains have been made in targeting glucagon-like peptide-1 receptors (a GPCR closely related to GCGR) but with the caveat of severe, adverse side-effects.[11] Encouraging results have recently come from Eli Lilly and Company who have been testing a small molecule antagonist of the GCGR (LY2409021) in phase two trials with some success, providing hope for some more specific control of diabetes mellitus.[10] In addition to diabetes mellitus, future development of signal bias modulators promise to provide focused therapies for obesity and heart disease, as well as related secondary pathological conditions such as hypertension and cancer.
See Also
PSI Structural Biology Database
G protein-coupled receptors
StructureSection load='4l6r' size='350' side='right' caption='Structure of the Class B Human Glucagon G Protein Coupled Receptor-PDB 4L6R' scene='72/721536/Class_b_gpcrs/4'
Human Glucagon Class B G Protein-Coupled Receptors (GPCRs)
Introduction
Human glucagon class B G protein-coupled receptors (GPCRs), also known as secretin-like receptors, are a subfamily of GPCRs and very similar in structure to the more well known class A (rhodopsin-like) glucagon receptor family. [13] Located in the liver, class B glucagon receptors (GCGRs) are activated by the binding of the hormonal peptide glucagon. Glucagon binding leads to the release of glucose into the bloodstream and plays an essential role in glucose homeostasis. Class B GCGRs are composed of a seven transmembrane domain (7TM) and an extracellular domain (ECD) that are required for glucagon binding.
Structures of Class A vs. Class B GPCRs
Class A vs. class B glucagon receptors share less than fifteen percent sequence homology, but both share a 7TM domain. [13] Understanding for class A family of GCGRs of the structure-function mechanism has made great progress over the past few years, but understanding of class B has fallen behind but is now catching up. [14] Comparison of the helices to that of the helices showed that the general orientation and positioning of the alpha helices are conserved through both classes. Detailed structural alignments of the two GPCR subclasses revealed multiple sequence misalignments in the transmembrane region signifying a variety of structural deviations in the transmembrane helices. [14] The N-terminal end of in class B GCGR, located in the 7TM, is longer than any known class A GPCR structure and stretches three supplementary helical turns above the extracellular (EC) membrane boundary. This region is referred to as the
. The stalk is involved in glucagon binding and helps in defining the orientation of the ECD with respect to the 7TM domain. [14] Also specific to class B GPCRs, a Gly residue at position 393 induces a ; this bend is stabilized by the hydrophobic interaction between the . One of the most distinguishable characteristics of the class B 7TM is the of 25 degrees and its length compared to that of , which is much shorter. This helical tilt results from Glu 406 in helix VIII that is fully conserved in secretin-like receptors and forms two interhelical salt bridges with conserved residues Arg 173 and Arg 346. [14] Despite these differences, a vital region that is conserved in both class B and class A receptors is the disulfide bond between in extracellular loop two (ECL2). This bond stabilizes the receptors entire 7TM fold. Lastly, the locations of the extracellular tips for class B glucagon receptors allow for a much wider and deeper ligand-binding pocket than any of the class A GPCRs. [14] These wide extracellular tip locations specifically occur between two sets of alpha helices, (Figure 1).
Figure 1: Extracellular tips of the 7TM helices. Helices two and six are shown in green, while helices three and seven are shown in red
How These Structures Lead to Function
Figure 2: Open conformation in contrast to the closed conformation. The movement of the single helix over the top of the transmembrane domain is the most distinguishable characteristic between closed and open conformation. The is not accessible to glucagon in the closed conformation.
[15] Structurally, the 7TM and its signature seven helical structure is involved in signaling via coupling to heterotrimeric G proteins that activate adenylate cyclase to increase the levels of intracellular cyclic AMP. Additionally, this coupling increases levels of IP3 and intracellular calcium levels. [14] The wider and deeper ligand-binding pocket of class B GPCRs allows for a vast array of molecules to be bound that in turn allow for numerous functions activated by peptide receptors. [16] The conformation and orientation of the 7TM and the ECD regions dictate the functionality of the class B G protein-coupled receptor, which has an open and closed conformation of the GCGR (Figure 2). The open conformation is when glucagon can bind to GCGR; in the closed conformation binding does not occur.[16]
Figure 3: Binding Pocket Residues: Residues with side chains of carbon(utilizing the
hydrophobic effect) are shown in green and side chains containing oxygen (
hydrophilic) are shown in red. The properties of hydrophobicity and hydrophilicity of the residues create the
binding affinity of glucagon.
[16] This image depicts the open conformation of GCGR.
The residues in the binding pocket that are in direct contact with the glucagon molecule are polar (utilizing the attraction of opposite charges or dipoles for glucagon binding) or are hydrophobic (utilizing the hydrophobic effect). The binding site location of the hormone peptide ligand has been identified, and the N-terminus of glucagon is known to bind partly with the ECD while the rest of glucagon binds deep into the binding pocket (Figure 3). The amino acids at the N-terminus of the class B 7TM have the ability to form hydrogen bonds and ionic interactions, which can be seen in the amino acid sequence of glucagon (Figure 4). [17]
Figure 4: Amino Acid Sequence of GlucagonThe primary sequence of glucagon, ligand to GCGR, is 29 amino acids.
Figure 5: Structure of Glucagon: The side chains of the residues making up glucagon are depicted. Coloration on the side chains indicate certain
atoms that determine the properties the residues hold. The blue indicates a
nitrogen atom (hydrophilic properties), the green on the side chains indicates carbon atoms (non-polar hydrophobic properties), and the red coloration indicates an
oxygen atom (hydrophilic properties).
PDB 1GCN
There are specific amino acid interactions that maximize affinity. This includes the alpha helical structure of the . The alpha helical structure of the stalk interacts directly with glucagon, as it extends nearly three helical turns above the membrane. When the alpha helix of the stalk is disrupted, the affinity of glucagon for GCGR decreases. A mutagenesis study mutating to a proline. Proline disrupts helices. The Ala135Pro mutant had significant lower affinity for glucagon.[14] Furthermore, there are certain interactions that hold the helices of the 7TM in the conformation that maximizes affinity. [16] The high affinity conformation of GCGR is the open conformation, when glucagon can bind. Without these specific interactions between the residues, the open conformation is not stabilized and GCGR remains in the closed conformation, where glucagon cannot bind. [14] The disulfide bond between serves to hold the helices in the proper orientation for binding and stabilizes the open conformation. Additionally, the salt bridges between
hold the open conformation together for higher affinity (Figure 6). [16]
Figure 6: Salt Bridge. The non-covalent interactions between residues form a
tridentate salt bridge. The Glu 406 acts as the central residue in the tridentate salt bridge; Arg 173 and Arg 436 both
chelating with Glu 406. The salt bridge is located on the intracellular side of the transmembrane helices.
Glucagon Signaling Pathway
Glucagon binds to the open conformation of GCGR; the GCGR located on the plasma membrane. Glucagon binding to GCGR induces a conformational change in GCGR. This conformation change induces the active state of the protein. The active state of the protein exchanges a guanosine diphosphate (GDP) for guanosine triphosphate (GTP) that is bound to the alpha subunit. With the GTP in place, the activated alpha subunit dissociates from the heterotrimeric G protein'sbeta and gamma subunits. Following dissociation, the alpha subunit can activate adenylate cyclase. Activated adenylate cyclase, catalyzes the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP). cAMP then serves as a secondary messenger to activate, through allosteric binding, cAMP dependent protein kinase A (PKA). PKA activates via phosphorylation the phosphorylase b kinase. The phosphorylase b kinase phosphorylates glycogen phosphorylase b to convert to the active form, phosphorylase a. Phosphorylase a finally catalyzes the release of glucose-1-phosphate into the bloodstream from glycogen polymers (Figure 7).
Figure 7: Metabolic Regulation of Glycogen by Glucagon.Depicted is the visualization of the glucagon signaling pathway through the GCGR. The location of the GCGR, the release of the alpha subunit from the beta and gamma subunits, and the enzyme cascade to result in the releasing of glucose are depicted. Abbreviations for the enzymes in the cascade include- PPK: phosphorylase kinase; PYG b: glycogen phosphorylase b; PYG a: glycogen phosphorylase a.
Clinical Relevancy
Of the fifteen human class B GPCRs, eight have been confirmed as potential drug target. [18] However, overall family B GPCRs have been difficult drug targets. This difficulty is partially related to the inherent flexibility in the class B GCGR 7TM. The flexibility comes from the ability of GCGR to be a receptor many ligands. The GCGR's interactions on the extracellular side of the receptor may provide evidence to how class B receptors adjust the conformational spectra for various ligands. Researchers hope to show how these conformations can be utilized in potential treatments of a wide array disorders. [18]
Potential Inhibitors for GCGR
There are potential class B GCGR inhibitors that have clinic relevancy. These inhibitors to class B GCGRs have primarily focused on allosteric inhibitors with high specificity and the ability to treat diseases including: stress disorders, managing hyperglycemia, and also alternative mechanisms for treating migraines. [19] Inhibitors include monoclonal antibodies which inhibit glucagon receptors through an allosteric mechanism. [20] There is further research still to be done on GCGR to determine more inhibitors for clinical relevance.
Class B Human Glucagon G-Protein Coupled Receptor
StructureSection load='4l6r' size='340' side='right' caption='Human Glucagon Class B GPCR ( 7tm PDB: 4l6r, ECD PDB: 4ers)' scene='72/721538/Glucagon_receptor/2'
Introduction
The human glucagon receptor (GCGR) is one of 15 secretin-like, or Class B, G-protein coupled receptors (GPCRs). Like other GPCRs, it has a helical domain and a globular N-terminus (ECD).
Function
The glucagon receptor plays an important role in glucose homeostasis. During times of fasting (or low blood sugar) the pancreas produces glucagon to activate the GCGR in the liver. The binding of glucagon to the extracellular side of GCGR leads to the activation of the receptor. On the intracellular side, upon this activation, a guanine diphosphate (GDP) is exchanged for a guanine triphosphate (GTP) - which, in turn, activates adenylate cyclase. Converting adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), adenylate cyclase initiates protein kinase A (PKA) activity, which releases glucose into the blood stream. Overall, activation of the GCGR elevates blood sugar levels.
Structure
Class B vs. Class A
Like all classes of glucagon receptors, which include class A (rhodopsin-like), B (secretin-like), and C (metabotropic glutamate), GCGR has a 7tm domain. While class B receptors do share characteristics with class C receptors, they are more similar to class A receptors. Class B receptors and class A receptors share less than 15% sequence homology; however, they do share similar signal transduction mechanisms as well as the 7tm domain. The orientations and positions of the 7tm helices are also conserved between both classes of glucagon receptors[21].
However, one particular difference between class A receptors and class B receptors is an inward shift of the intracellular component of Helix VII. In class A receptors this inward shift is instrumental in receptor activation, yet in class B receptors it remains unclear what role this shift plays[21].
In contrast to class A glucagon receptors which have a proline kink, in all secretin-like class B glucagon receptors a Glycine at position 393 in Helix VII allows for a . This glycine helical bend is fully conserved in all secretin-like class B receptors.
Another important structural component found in all secretin-like class B receptors are the two conserved salt bridges found between Arg 346 and Glu 406 and Arg 173 and Glu 406. These are a distinct feature of class B receptors because their interaction results in the distinct stalk found only in class B receptors[21].
While interface interactions between helices VI, V, and III, are not unique to class B receptors because certain homologous and even conserved residues exist in class A receptors (like Tyr 239 and Leu 358), as a part of the interface between helices VI, V, and III, a Class B-specific hydrogen bond occurs between Asn 318 of Helix V and Leu 242 of Helix III [21].
GCGR-Specific Traits
Helix I Stalk Region
The tip of Helix I extends above the cell membrane into the extracellular space creating a . This region is longer than any other class of GPCR and extends three α-helical turns above the plane of the membrane. It is proposed that the stalk helps to capture the glucagon peptide and facilitates it's insertion into the 7tm[21].
Intracellular Helix VIII
The GCGR also contains an intracellular Helix VIII that is comprised of roughly 20 amino acids at the C-terminal end. This helix tilts approximately 25 degrees away from the membrane - the corresponding position in class A receptors are turned toward the membrane[21]. Although researchers are not entirely sure of its function, this helix is completely conserved in class B structures.
Binding Pocket
Figure 1. GCGR Binding Pocket. A cross-section of the GCGR binding pocket shows its width and depth
The class B GPCR has the widest and longest of all other classes of GPCRs. The distance between the EC tips of Helicies II and VI as well as between the tips of Helicies III and VII are some of the largest among the GPCRs
[21]. As a result, the
binding cavity of GCGR is located deeper inside the receptor, meaning glucagon binds much closer to the cell membrane.
Other Unique Structural Features
An important interface stabilization interaction between Helices I and VII occurs between Ser 152 of Helix I and Ser 390 of Helix VII. Due to their close proximity to one another, they form an important which stabilizes the structure of GCGR. Mutations to the homologous residues Ser 135 and Ser 392 have been shown to alter receptor signaling in glucagon-like peptide-1 receptor (GLP1R).
Glucagon Binding
Figure 2. Bound Molecule of Glucagon. A molecule of glucagon is shown bound to the GCGR's ECD (shown in magenta)
Research has shown that class B GCPRs exist in either an
open or closed conformation differentiating between the receptor's active and inactive states. The active, or open conformation, is characterized by an intracellular outward movement of (breaking hydrogen bonds between and )
[22] and an extracellular rotation of the ECD until it is almost perpendicular to the membrane surface
[23]. While the stalk region of Helix I helps to facilitate the motion of the ECD, intracellular G-protein coupling and extracellular glucagon binding stabilized this active state. In the abscence of glucagon, however, the GCGR adopts a closed conformation in which all three of the extracellular loops of the 7tm () can interact with the ECD
[23]. In this closed state, the ECD covers the extracellular surface of the 7tm. To transition between states, the ECD rotates and moves down towards the 7tm domain. This transition mechanism is consistent with the "two-domain" binding mechanism of class B GCPRs in which (1) the C-terminus of the ligand first binds to the ECD allowing (2) the N-terminus of the ligand to interact with the 7tm and activate the protein
[22].
Clinical Relevance
Because of GCGR's role in glucose homeostasis, GCGRis a potential drug target for Type 2 diabetes. Specifically, molecules that antagonize the glucagon receptor may be able to lower blood sugar levels. Among experimental treatments, two antibodies, mAb1 and mAb23, target the ECD domain of the GCGR interrupting glucagon binding[24]. While the entire cleft of the ECD is blocked by mAb1, mAb3 blocks glucagon binding by stabilizing a conformation of the ECD that promotes receptor inactivation [24]. Another antibody, mAb7, inhibits GCGR allosterically[25]. Through binding to a site outside of the binding pocket, mAb7 inhibits the receptor without interacting with essential glucagon binding residues. Disrupting the normal interactions between the ECD and the 7tm domains, these antibodies inhibit the receptor's function and help to lower blood glucose level.
As GCGR holds great promise as a therapeutic target, there are currently three drugs under development that are designed to treat Type 2 Diabetes by targeting the human glucagon receptor[22]. Although they are still in phase I of their clinical trials, preliminary research has shown promising results[26].
