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spinqualitylabelsall models Structure of the Class B Human Glucagon G Protein Coupled Receptor-PDB 4L6R Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Human Glucagon Class B G Protein-Coupled Receptors (GPCRs) Introduction File:Screen Shot 2016-03-29 at 12.45.02 PM.png Figure 1: Glucagon, shown in green, bound to its receptor in the class B family Human glucagon class B G protein-coupled receptors (GPCRs), also known as secretin-like receptors, are a subfamily GPCRs and very similar in structure to the more well known class A (rhodopsin-like) glucagon receptor family. Located in the liver, class B glucagon receptors (GCGRs) are activated by the binding of the hormonal peptide glucagon (Figure 1). 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 extracellular domain (ECD) that are required for glucagon binding. Structures of Class A vs. Class B GPCRs In comparison, class A vs. class B glucagon receptors share less than fifteen percent sequence homology, but both share this 7TM domain. Understanding for class A family of GCGRs for the structure-function mechanism has made great progress over the past few years, but understanding of class B has fallen behind but has recently started catching up. Comparison of the Class B GCGR 7TM to that of a class A 7TM 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 gaps in the transmembrane region signifying a variety of structural deviations in transmembrane helices. Also specific to class B GPCRs, a Gly residue at position 393 induces a bend in helix VII; this bend is stabilized by the hydrophobic interaction between the glycine 393 and phenylalanine 184. One of the most distinguishable characteristics of the class B 7TM is the helix VIII tilt of 25 degrees compared to that of class A, which has no tilt. 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. Despite these differences, a vital region that is conserved in both class B and class A receptors is the disulfide bond between Cys 294 and Cys 224 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. These wide extracellular tip locations specifically occur between two sets of alpha helices, (Figure 2). Figure 2: 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 Structurally, the N-terminal extracellular domain (ECD) and the 7TM comprise the signature seven helical structure that 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 inositol phosphate and intracellular calcium levels. The wider and deeper ligand-binding pocket of class B GPCRs allows for a vast array of receptors to be bound that allow for numerous functions activated by peptide receptors. The conformation and orientation of the 7TM and the ECD regions dictate the functionality of the protein, which has an open and closed conformation of the GCGR. When glucagon binds to GCGR, the open conformation of GCGR is stabilized. There is no clear binding site location of the hormone peptide ligand, but they do know the N-terminus of glucagon binds deep into the binding pocket. The amino acids at the N-terminus have the ability to form hydrogen bonds and ionic interactions involved, which can be seen in the amino acid sequence of glucagon (Figure 3). Figure 3: Amino Acid Sequence of Glucagon Many of the residues that are in direct contact with the glucagon molecule are charged or are polar. Figure 4: Salt Bridge. The salt bridge is located on the intracellular side at the bottom of the protein, in relation to the orientation it holds within the cell membrane. It is made between residues Glu 406, Arg 173, and Arg 346, as labeled in the figure. There are also many smaller residues on glucagon that support the bulky residues on the GCGR. These residues are located within the binding pocket of the 7TM. There are specific amino acid interactions that hold the helices of the 7TM in the closed conformation that maximizes affinity. This includes the disulfide bond between Cys 294 and Cys 224 that was mentioned earlier that serves to hold the ECL1 and ECL2 in the proper orientation. Additionally, the salt bridges between Glu 406, Arg 173, and Arg 346, also mentioned earlier, hold the conformation together for higher affinity. Finally, alpha helical structure of the stalk is imperative to the affinity and binding of the glucagon. Figure 5: Binding Pocket Residues: side chains of carbon chains are shown in green and side chains containing oxygen are shown in red Glucagon Signaling Pathway For glucagon to initiate the breakdown of glycogen into glucose, it follows a specific signaling mechanism (Figure 6). Glucagon binds to a G protein-coupled receptor located on the plasma membrane, and this binding induces a conformational change that activates G proteins which exchanges a guanosine diphosphate (GDP) for guanosine triphosphate (GTP) that is bound to the alpha subunit. Now with the GTP in place, the activated alpha subunit leaves the heterotrimeric G protein and activatesthe next enzyme in the cascade, adenylate cyclase. Once adenylate cyclase is activated, it catalyzes the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP). This in turn activates protein kinase A (PKA) that then activates phosphorylase kinase. Finally, the phosphorylase kinase phosphorylates glycogen phosphorylase b that is then turned into its activated form, glycogen phosphorylase a. This phosphorylase A enzyme is responsible for catalyzing the release of glucose-1-phosphate into the bloodstream from glycogen polymers. Figure 6: Glucagon Signaling Pathway Clinical Relevancy Of the fifteen human class B GPCRs, eight have been identified as potential drug target. Therapeutic agents have been created from the peptides themselves within this protein, but overall pharmaceutical companies have had difficulty creating agents that act on family B GPCRS. There is an outward appearance and inherent flexibility in the class B GCGR 7TM because of conserved hydrogen bonds that flank a glycine residue, and this structure along with the ECD and its role of interactions on the extracellular side of receptors may provide evidence to how class B receptors adjust its conformational spectra for various receptors. Researchers hope to show how these conformations can be utilized in potential treatments of a wide array disorders. Potential Inhibitors Research for class B GCGR inhibitors is primarily looking into allosteric inhibitors having the ability to target specific receptors in order to treat problems like stress disorders, managing hyperglycemia, and also alternative mechanisms for treating migraines. Known inhibitors include monoclonal antibodies which inhibit glucagon receptors through an allosteric mechanism. The monoclonal antibodies bind to two different sites, the ECD opposite of the binding region and then the helical portion of the ECD as well. Research Determining the structure of class B GCGRs is a reason for its lack of advanced knowledge in the field, but X-ray crystallography and NMR have been the main processes performed and have had some success with it over the past couple years . X-ray crystallography displayed the crystal structure of ECDs of class B GPCRs in complex with their ligands along with the crystal structure of the 7TM. In addition to this, NMR has allowed the ability to directly understand structures of soluble amino-terminal domains of numerous members of the secretin-like family that bind peptide hormones. Primary sequences analysis have led to the finding of seven segments of eighteen or more relatively hydrophobic residues that are believed to represent transmembrane helices that take part in creating an intramembranous helical bundle . Also, mutagenesis has been used to determine which residues were necessary in maximizing affinity for glucagon. Finally, the orientation and mechanism of the peptide interactions within these structures are studied using peptide structure-activity relationships (SAR), receptor and ligand fragments, chimeric receptors, site-directed mutagenesis, photochemical cross-linking, and molecular modeling .