Human G-protein-coupled glucagon receptor (4L6R) from Homo sapiens
Created by: Alex Yahanda
The Human G-protein-coupled glucagon receptor (GCGR; PDB ID: 4L6R) from Homo sapiens is one of 15 members of the class B G protein-coupled receptor (GPCR) family. The GPCR family is a group of receptor proteins that facilitates cellular responses upon binding of a ligand. The GCGR primarily affects glucose production by the liver through binding to glucagon (PDB ID: 1GCN), a 29 amino acid hormonal peptide that is secreted from pancreatic alpha-cells near the Islets of Langerhans (1). Glucagon receptors are also located in cells of the brain, intestine, kidneys, heart, and adipose tissue. Glucagon is secreted during times of fasting or low blood glucose levels. Glucagon secretion ultimately results in the inactivation of insulin and the release of glucose from the liver into the bloodstream (2). To that end, glucagon and the GCGR play vital roles in maintaining glucose homeostasis. Moreover, the GCGR is now being researched for its role in type 2 (insulin independent) diabetes (2, 3). Type 2 diabetics tend to struggle with excess glucose production, which may be closely linked to glucagon receptors.
Binding of glucagon to the GCGR, as with the binding of other ligands to their respective GPCRs, leads to the activation of other G proteins such as Gs and Gq (2). Activation of those G proteins results in the initiation of adenylyl cyclase activity and increased intracellular levels of cAMP, which subsequently leads to increased levels of protein kinase A and phosphoinositol turnover. The transmitted signals tell the liver to initiate the production and release of glucose through glycogenolysis and gluconeogenesis. At this point, glycogenesis is also inhibited (1-5).
The human GCGR is a 477-amino acid membrane protein consisting of one subunit. In other animals, the number of amino acids in glucagon receptors may vary; in rats, for instance, the GCGR is 485 amino acids long (1). The secondary structure of the GCGR is predominantly composed of alpha helices and random coils. The GCGR is well embedded in the cell membrane via seven transmembrane alpha helices. Since these alpha helices span the hydrophobic cell membrane interior, they contain mostly hydrophobic residues. Parts of the GCGR that are exposed to intra- or extracellular domains contain more polar, acidic, and basic residues (6). In all GCGRs, there are four N-linked glycosylation spots and a motif in the third intracellular loop that is essential for receptor protein activation. Crystallization of the GCGR indicates that its extracellular N-terminus domain is particularly long for class B GPCRs and contains more residues than any corresponding domain in class A GPCRs (6). This extracellular domain, termed the “stalk,” is probably useful in glucagon binding and orienting the ligand with respect to the protein binding site. The primary structure of the stalk includes the proper residues for recognizing and initiating binding with glucagon (6).
There are a number of structurally and functionally important residues in the GCGR. A disulphide bond between Cys-224 and Cys-294 stabilizes the transmembrane folds (6). Additionally, transmembrane helices I and II stabilize each other via hydrophobic interactions between Leu-156 and Phe-184. More helix stabilization occurs between helices I and VII by way of hydrogen bonds between Ser-152 and Ser-390. Glu-406 in helix VIII (an intracellular alpha helix) interacts with Arg-173 and Arg-346 to form two salt bridges, which creates an ionic network specific to class B receptors. Another bond specific to class B receptors is formed between helices III and V through interhelical hydrogen bonds between Asn-318 and Leu-242 (6).
The complete mechanism whereby glucagon binds to the GCGR is not perfectly understood. It is thought that the extracellular stalk region connected to helix I of the GCGR first binds the C-terminus of glucagon. This hypothesis is supported by the fact that mutations leading to structural abnormalities in the stalk decrease the binding affinity between the GCGR and glucagon (6). This C-terminus binding leads to glucagon’s N-terminus having an increased affinity for binding with a cleft formed by the GCGR’s transmembrane helices (4). The full binding of glucagon to the GCGR induces a conformational change in the intracellular protein domain, which activates the protein and initiates the signal cascade (5). Asp-63, Tyr-65, and Lys-98 are especially helpful in stabilizing the receptor’s extracellular domain during glucagon binding. Trp-36 is an important site for hydrophobic interactions with glucagon’s C-terminal side. More hydrophobic interactions likely occur between glucagon’s Phe-6 and the GCGR’s Gln-142, as well as glucagon’s Tyr-10 and the GCGR’s Tyr-138 (6). The precise glucagon binding site among the GCGR’s seven transmembrane helices is not exactly known. But, it is now believed that the first five alpha helices exhibit a flexibility that places the glucagon binding pocket deep within the transmembrane domain. Glucagon thus binds in closer proximity to the cell membrane than previously expected. Glucagon was thought to bind near the top of the transmembrane helices’ extracellular domains, further above from the cell membrane. This proposition is also supported by mutagenesis studies. Mutations in the residues that are within the hypothesized binding pocket result in a greatly reduced binding affinity between glucagon and the GCGR (6).
Since the GCGR may be closely tied to type 2 diabetes, the effects of blocking the receptor and the potential usefulness of GCGR-inhibiting drugs are currently the subjects of much investigation. Type 2 diabetes involves increased glucose production, pancreatic B-cell malfunction, and insulin insensitivity. Research has indicated that elevated glucagon levels may be a contributing factor to the increased blood glucose levels found in type 2 diabetics (3). Inhibiting interactions between glucagon and the GCGR, then, may help slow excess glucose production and result in better control of blood glucose levels. In the past, researchers have tried to inhibit glucagon receptors via small-molecule approaches. More recently, antibodies have been used to develop better methods for interfering with glucagon-GCGR interactions. For instance, it was demonstrated that certain antibodies that target the GCGR’s extracellular domain could inhibit glucagon binding (4). By using monoclonal antibodies mAb1 and mAb23, a GCGR-antibody complex can be formed (PDB ID: 4ERS) that reduces interactions between glucagon and the GCGR (4). These antibodies interfere with the GCGR’s extracellular domain, blocking the crucial interactions that occur at the extracellular domain prior to glucagon binding. In a similar study, different antibodies were used to antagonize glucagon receptors (3). A promising immunological approach to blocking glucagon receptors (no PDB ID for the structure) was developed using monoclonal antibody mAb. Indeed, in those experiments, mAb exhibited a higher affinity for GCGR than glucagon, demonstrating its potential effectiveness at blocking receptors.
The GCGR is a member of the G protein superfamily. More specifically, it is part of the class B G protein-coupled receptor family. It has an isoelectric point of 9.01 and a weight of 54,009.9 Da (7). Concerning primary structure, a NCBI protein Blast search indicates that the GCGR is very similar to the vasoactive intestinal peptide receptors (E=4e-180; no PDB IDs found) and secretin receptors (E=1e-180; no PDB IDs found) found in both Homo sapiens and many other species. Protein Blast is a server that compares proteins by their primary structures (8). Primary structure similarity between those three proteins is not surprising. The GCGR, vasoactive intestinal peptide receptors, and secretin receptors are all GPCR with seven transmembrane alpha helices (1). Comparable amino acid sequences are expected, as the proteins have similar secondary structures and functions. Expasy, a bioinformatics server run by the Swiss Institute of Bioinformatics, further confirms sequence and structural homology between the GCGR and other membrane receptors found in Homo sapiens such as the glucagon-like peptide receptors (PDB ID: 3IOL for the extracellular domain) (9). The GCGR is most similar in tertiary structure to other class B GPCRs, especially chain A of the corticotropin-releasing factor receptor 1 (CRF1R; PDB ID: 4K5Y), as shown by DALI (Z=21.8). DALI is a server that compares proteins by their tertiary structures using a sum-of-pairs method (10). Chain A of the CRF1R, according to DALI, is the protein with the closest three-dimensional structure to the GCGR. It was thus chosen as a comparison protein. As is characteristic of GPCRs, the CRF1R has seven transmembrane helices and an N-terminus extracellular domain. The CRF1R operates in a similar manner to the GCGR, but with a different ligand. The CRF1R binds corticotropin-releasing factor (CRF; PDB ID: 1GO9), a 41-amino acid neuropeptide with a helical structure quite similar to glucagon. The extracellular domain of the CRF1R is not as long as the stalk region of the GCGR. But, like the GCGR, the extracellular domain interacts with the C-terminus of the receptor’s peptide ligand, increasing the binding affinity of the peptide’s N-terminus (11). Binding of CRF leads to conformational changes in the G proteins and initiates an adenylyl cyclase signaling pathway. The pathway ultimately leads cells to release hormones that are likely connected to stress or anxiety. Thus, drug developers are currently examining the CRF1R. Finding ways to successfully inhibit the CRF1R could lead to drugs that combat depression and other debilitating mental conditions (11).