[[Image:ECD_bound_to_glucagon.png|200 px|left|thumb|'''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 [http://www.nature.com/ncomms/2015/150731/ncomms8859/fig_tab/ncomms8859_F3.html 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 <scene name='72/721538/Helix_v_and_vi/1'>helicies V and VI</scene> (breaking hydrogen bonds between <scene name='72/721538/Arg173-ser350_h_bond/1'>Arg173-Ser350</scene> and <scene name='72/721538/Arg173-ser350_h_bond/2'>Glu245-Thr351</scene>) <ref>DOI 10.1039/C6CP00798H</ref> and an extracellular rotation of the ECD until it is almost perpendicular to the membrane surface <ref name ='conformation_article'>PMID:26227798 </ref>. 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 (<scene name='72/721538/Ecls/1'>ECL1, ECL2, and ECL3</scene>) can interact with the ECD ''citation needed''. 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 ''citation needed''.
[[Image:ECD_bound_to_glucagon.png|200 px|left|thumb|'''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 [http://www.nature.com/ncomms/2015/150731/ncomms8859/fig_tab/ncomms8859_F3.html 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 <scene name='72/721538/Helix_v_and_vi/1'>helicies V and VI</scene> (breaking hydrogen bonds between <scene name='72/721538/Arg173-ser350_h_bond/1'>Arg173-Ser350</scene> and <scene name='72/721538/Arg173-ser350_h_bond/2'>Glu245-Thr351</scene>) <ref>DOI 10.1039/C6CP00798H</ref> and an extracellular rotation of the ECD until it is almost perpendicular to the membrane surface <ref name ='conformation_article'>PMID:26227798 </ref>. 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 (<scene name='72/721538/Ecls/1'>ECL1, ECL2, and ECL3</scene>) can interact with the ECD <ref name ='conformation_article'>PMID:26227798 </ref>. 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 ''citation needed''.
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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 stimulates gluconeogenesis, through adenylate cyclase that initiates protein kinase A (PKA) activity[1]. This pathway synthesizes glucose, elevating 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[2].
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[2].
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
Glu 406 Salt Bridges
. These salt bridges are a distinct feature of class B receptors only because their interaction results in the distinct stalk found only in class B receptors[2].
As a part of the interface stabilization 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.
GCGR-Specific Traits
Helix I Stalk Region
The tip of Helix I extends above the cell membrane into the extracellular space creating a stalk region. 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[2].
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[2]. 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 binding pocket 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[2]. 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 hydrogen bond which stabilizes the structure of GCGR.
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 helicies V and VI (breaking hydrogen bonds between Arg173-Ser350 and Glu245-Thr351) [3] and an extracellular rotation of the ECD until it is almost perpendicular to the membrane surface [4]. 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 (ECL1, ECL2, and ECL3) can interact with the ECD [4]. 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 citation needed.
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[5]. 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 citation needed. Another antibody, mAb7, inhibits GCGR allosterically[6]. 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.
↑Lotfy M, Kalasz H, Szalai G, Singh J, Adeghate E. Recent Progress in the Use of Glucagon and Glucagon Receptor Antago-nists in the Treatment of Diabetes Mellitus. Open Med Chem J. 2014 Dec 31;8:28-35. doi: 10.2174/1874104501408010028., eCollection 2014. PMID:25674162 doi:https://dx.doi.org/10.2174/1874104501408010028
↑ 2.02.12.22.32.42.5Siu FY, He M, de Graaf C, Han GW, Yang D, Zhang Z, Zhou C, Xu Q, Wacker D, Joseph JS, Liu W, Lau J, Cherezov V, Katritch V, Wang MW, Stevens RC. Structure of the human glucagon class B G-protein-coupled receptor. Nature. 2013 Jul 25;499(7459):444-9. doi: 10.1038/nature12393. Epub 2013 Jul 17. PMID:23863937 doi:10.1038/nature12393
↑Li Y, Sun J, Li D, Lin J. Activation and conformational dynamics of a class B G-protein-coupled glucagon receptor. Phys Chem Chem Phys. 2016 Apr 20. PMID:27094704 doi:https://dx.doi.org/10.1039/c6cp00798h
↑ 4.04.1Yang L, Yang D, de Graaf C, Moeller A, West GM, Dharmarajan V, Wang C, Siu FY, Song G, Reedtz-Runge S, Pascal BD, Wu B, Potter CS, Zhou H, Griffin PR, Carragher B, Yang H, Wang MW, Stevens RC, Jiang H. Conformational states of the full-length glucagon receptor. Nat Commun. 2015 Jul 31;6:7859. doi: 10.1038/ncomms8859. PMID:26227798 doi:https://dx.doi.org/10.1038/ncomms8859
↑Koth CM, Murray JM, Mukund S, Madjidi A, Minn A, Clarke HJ, Wong T, Chiang V, Luis E, Estevez A, Rondon J, Zhang Y, Hotzel I, Allan BB. Molecular basis for negative regulation of the glucagon receptor. Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14393-8. Epub 2012 Aug 20. PMID:22908259 doi:https://dx.doi.org/10.1073/pnas.1206734109
↑Mukund S, Shang Y, Clarke HJ, Madjidi A, Corn JE, Kates L, Kolumam G, Chiang V, Luis E, Murray J, Zhang Y, Hotzel I, Koth CM, Allan BB. Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor. J Biol Chem. 2013 Nov 4. PMID:24189067 doi:https://dx.doi.org/10.1074/jbc.M113.496984
