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G protein coupled receptors (GPCRs) are recognized as the largest known class of integral membrane proteins and are divided into five families; the rhodopsin family (class A), the secretin family (class B), the adhesion family, the glutamate family (class C), and the frizzled/taste family (class F). 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 response. The variants all contain a similar seven α-helical transmembrane domain (TMD or 7TMD) that, once bound to its peptide ligand, undergoes conformational change and tranduces a signal to coupled, heterotrimeric G proteins which initiate intracellular signal pathways and generate physiological and pathological processes. [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 concentration of cAMP, inositol phosphate, and calcium levels in cyto. [2] These signals 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 to companies developing novel molecules. [3] Structurally based approaches to the development of small-molecule agonists and antagonists have been hampered by the lack of accurate Class B TMD visualizations until recent crystal structures of corticoptropin-releasing factor receptor 1 and human glucagon were realized. [4] [5]

The glucagon class B GPCR (GCGR) is involved in glucose homeostasis through the binding of the signal peptide glucagon.


7TM structure of human class B GPCR 4L6R

Drag the structure with the mouse to rotate


Contents

Introduction

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. Once the peptide hormone is released, it binds to GCGR which is a 485 amino acid protein found in the liver, kidney, intestinal smooth muscle, brain, and adipose tissues. [6] Upon binding, signaling is initiated to heterotrimeric G-proteins containing Gαs. [7] Additionally, GCGR can regulate additional signal pathways including G-proteins of the Gαi family through the adoption of differing receptor conformations. [8]


Structural Considerations

Comparison between Class A and Class B GPCRs

The class B GPCRs, of which GCGR is a member, are different from other Class A GPCRs in several ways. The first is that class B GPCRs contain a protrusion known as a 'stalk,' which is a three α-helical turn elongation of the N-terminus that protrudes past the extracellular (EC) membrane. Structural integrity of this domain in GCGR is essential to ligand binding affinity. (Fig's 1 and 2)
Fig. 1: A135P Mutation and effect on stalk stability .
Fig. 1: A135P Mutation and effect on stalk stability [5].
Fig. 2: Stalk stabilized by salt bridge between Glu133-Lys136. Residues in yellow are demonstrated to have an effect on ligand binding affinity.
Fig. 2: Stalk stabilized by salt bridge between Glu133-Lys136. Residues in yellow are demonstrated to have an effect on ligand binding affinity.[5]








Secondly, the extracellular loop 1 (ECL1) is 3-4 times longer than comparable loops in class A GPCRs, and also affects ligand binding affinity. (Fig. 3)[5]
Fig. 3: Active sites linked to glucagon binding affinity located on ECL1 are labeled.
Fig. 3: Active sites linked to glucagon binding affinity located on ECL1 are labeled[5].








Most notably, class B GPCRs contain a prominent central splay (Fig. 4) which is solvent filled and accessible from the extracellular side. This central splay is notably absent from class A GPCRs (Fig. 5) , represents a tantalizing target for agonists/antagonists, and is the focus of much current research into GCGR signal regulation. [3]

Fig. 4: Corticotropin-releasing factor 1 and glucagon receptors; Class B GPCRs with notable central splay
Fig. 4: Corticotropin-releasing factor 1 and glucagon receptors; Class B GPCRs with notable central splay
Fig. 5: Beta 2-adrenergic (class A) and glucagon receptors; showing an absence of central splay in Class A GPCRs.
Fig. 5: Beta 2-adrenergic (class A) and glucagon receptors; showing an absence of central splay in Class A GPCRs.












Structurally Significant GCGR 7TDM Residues

Fig. 6: Snake Plot of GCGR TMD
Fig. 6: Snake Plot of GCGR TMD[5]




The snake plot (Fig. 6) shows the conservation and effects of mutagenesis in the 7TMD structure of class B human GPCR. The highly conserved amino acids imply an importance to that functioning of the individual residues and their interactions. The amino acids which have a great impact on the function of the receptor are highlighted in teal, yellow, and black, and offer evidence that the position and interaction of the amino acid is crucial for protein function. Most of the residues that play an important role in glucagon binding face the main cavity of the 7TM structure. Mutagenesis in these positions highly compromises the functioning of the glucagon binding.


Functions of Glucagon receptor (GCGR)

Peptide binding and selectivity

It has been discovered that the large, soluble N-terminal extracellular domains (ECD) of GCGR are primary in ligand selectivity with the deep, ligand pocket (Fig. 7) of the TMD providing secondary recognition. [6]
Fig. 7: Active site buried deep in 7TMD of glucagon receptor.
Fig. 7: Active site buried deep in 7TMD of glucagon receptor.





Conformational changes

Because of the difficulty of stabilizing and crystallizing Class B TMDs, very little is known about the conformational changes that transduce cell signals endogenously. It is known that GCGR can regulate additional signal pathways including G-proteins of the Gαi family through the adoption of differing receptor conformations. Research is ongoing. [8]


Signaling pathways

GCGR generates downstream signals predominantly through the increase of intracellular cAMP, however there are other pathways being uncovered that are the result of GCGR adopting multiple, active conformations. Researchers are currently investigating how receptor activity-modifying proteins (RAMPs) interact with the ligand and GCGR in which the signaling bias of the receptor is altered. [9]


Kinetics

GPCR activity is regularly quantified by ligand binding affinity, potency, efficacy, and kinetics. These measurement are used to measure drug ligand interactions in vivo. Recently, GPCRs have been crystallized and catalogued, which tend to include a need to stabilize the receptor, emphasizing the instability of the G coupled protein receptor. Zhang et. al. imply the importance of receptor folding in the cell membrane, in the human class B GPCR the 7TM portion, for receptor stability and function. [10]


The Signal Peptide: Glucagon

Biological function of glucagon

Glucagon, a signaling ligand in the metabolic pathway, has three main biological functions.

Glucagon is 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. Once HMG-CoA has been phosphorylated, it is inactivated and cholesterol production is moderated to conserve energy.

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. 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.

Glucagon's main role is the regulation of blood glucose levels. 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.

[11]


Glucagon Peptide Stability and Active Binding Sites

Essential, conserved residues of glucagon, as discovered through mutagenesis and photo cross-linking studies have been labeled and colored in red. [5]


Active binding domains/sites

Through mutagenesis and photo-crosslinking studies, several residues deep within the central cavity of the GCGR 7TMD were discovered neighboring Glu362, which is approximately 19 angstroms from the base of the EC stalk and the location of Tyr138. (Fig. 8)

Fig.8: Relationship between Tyr138 and Glu362 - residues found to have direct relationship to glucagon binding affinity.
Fig.8: Relationship between Tyr138 and Glu362 - residues found to have direct relationship to glucagon binding affinity.

Four essential residues exist deep within the central cavity which all play strong roles in ligand binding affinity. (Fig. 9)

Fig. 9: Location of anchoring pocket within central cavity.
Fig. 9: Location of anchoring pocket within central cavity.[5]









A narrow entry gives way to a large, anchoring site for residues 1-4 of glucagon. (Fig. 10)

Fig. 10: Ballooned pocket functioning as anchoring site for glucagon residues 1-4.
Fig. 10: Ballooned pocket functioning as anchoring site for glucagon residues 1-4.

Essential to glucagon's binding, a long, N-terminal tail winds to a clump of 4 residues, culminating in bulge that fits into the central, anchoring site of the 7TMD. (Fig. 11)

Fig. 11: Surface visualization of glucagon demonstrating three dimensional fit of N-terminal tail into binding site of GCGR central cavity active site
Fig. 11: Surface visualization of glucagon demonstrating three dimensional fit of N-terminal tail into binding site of GCGR central cavity active site









Clinical relevance

Class B secretin-like receptors have gained relevance in therapeutics and drug targets. Maintaining information about the class B GPCRs conformational flexibility, allows for a better understanding of the receptor-ligand binding and its pharmaceutical relevance. The 7TM structure offers a direct connect between the extracellular and intracellular region, which offers a mechanism for signal transduction within the cell. GPCRs regulate cellular processes as required by the organs in which they are located. GPCR’s are used in the functioning of neuron synapses, ion transport regulation, homeostasis, cell division, and cell morphology. Mutations in the GPCR have been linked with retinitis pigmentosa, female infertility, nephrogenic diabetes insipidus, and familial exudative vitreoretinopathy. [12]


Future research direction

Research for Class A GPCRs is much more extensive than for its secretin, class B counterparts, although class B is proving to be a worthwhile to invest researching. The challenge of class B stabilization, expression, and molecular size , has made class B GPCRs particularly hard to assay. Biochemical research has increased in the class B specifications, because it has been realized that receptors can be modulated by more than the agonist and antagonists present in vivo. Leading research consists of a complex interwoven scheme of equilibria manipulation in multi-receptor conformations. [12]

Current drug targets

A variety of small molecule modulators have been developed over the past several years providing the promise of enhanced pharmaceutical regulation of GCGR. [6](Fig's. 12 and 13)

Fig. 12: Small molecule regulators of GCGR, part 1.
Fig. 12: Small molecule regulators of GCGR, part 1[6].
Fig. 13: Small molecule regulators of GCGR, part 2.
Fig. 13: Small molecule regulators of GCGR, part 2[6].












Possible structural considerations for large molecule agonists/antagonists

Utilizing the visualizations of the GCGR 7TMD and glucagon peptide ligand, dimensional/structural analyses can be performed to develop models for novel molecules of increasing specificity for GCGR binding/regulation. Performing a dimensional analysis between the binding pocket and the base of the EC stalk, a large pseudopeptide molecule of 17-24 angstroms in size could be utilized to mimic the characteristics of GCGR's natural ligand, glucagon. (Fig's. 14 and 15)

Fig. 14: Distance measurement of GCGR 7TMD Y138-D362 of 19-20 angstroms and labeled with complimentary glucagon interaction residues.
Fig. 14: Distance measurement of GCGR 7TMD Y138-D362 of 19-20 angstroms and labeled with complimentary glucagon interaction residues.
Fig. 15: Distance measurement of H1-Y10 of 22-24 angstroms and labeled with complimentary GCGR 7TMD residue interactions.
Fig. 15: Distance measurement of H1-Y10 of 22-24 angstroms and labeled with complimentary GCGR 7TMD residue interactions.



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References

  1. ↑ Zhang Y, Devries ME, Skolnick J. Structure modeling of all identified G protein-coupled receptors in the human genome. PLoS Comput Biol. 2006 Feb;2(2):e13. Epub 2006 Feb 17. PMID:16485037 doi:http://dx.doi.org/10.1371/journal.pcbi.0020013
  2. ↑ Bortolato A, Dore AS, Hollenstein K, Tehan BG, Mason JS, Marshall FH. Structure of Class B GPCRs: new horizons for drug discovery. Br J Pharmacol. 2014 Jul;171(13):3132-45. doi: 10.1111/bph.12689. PMID:24628305 doi:http://dx.doi.org/10.1111/bph.12689
  3. ↑ 3.0 3.1 Hollenstein K, de Graaf C, Bortolato A, Wang MW, Marshall FH, Stevens RC. Insights into the structure of class B GPCRs. Trends Pharmacol Sci. 2014 Jan;35(1):12-22. doi: 10.1016/j.tips.2013.11.001. Epub, 2013 Dec 18. PMID:24359917 doi:http://dx.doi.org/10.1016/j.tips.2013.11.001
  4. ↑ Hollenstein K, Kean J, Bortolato A, Cheng RK, Dore AS, Jazayeri A, Cooke RM, Weir M, Marshall FH. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature. 2013 Jul 25;499(7459):438-43. doi: 10.1038/nature12357. Epub 2013 Jul 17. PMID:23863939 doi:http://dx.doi.org/10.1038/nature12357
  5. ↑ 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 Siu 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
  6. ↑ 6.0 6.1 6.2 6.3 6.4 Yang DH, Zhou CH, Liu Q, Wang MW. Landmark studies on the glucagon subfamily of GPCRs: from small molecule modulators to a crystal structure. Acta Pharmacol Sin. 2015 Sep;36(9):1033-42. doi: 10.1038/aps.2015.78. Epub 2015, Aug 17. PMID:26279155 doi:http://dx.doi.org/10.1038/aps.2015.78
  7. ↑ Ahren B. Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes. Nat Rev Drug Discov. 2009 May;8(5):369-85. doi: 10.1038/nrd2782. Epub 2009 Apr, 14. PMID:19365392 doi:http://dx.doi.org/10.1038/nrd2782
  8. ↑ 8.0 8.1 Xu Y, Xie X. Glucagon receptor mediates calcium signaling by coupling to G alpha q/11 and G alpha i/o in HEK293 cells. J Recept Signal Transduct Res. 2009 Dec;29(6):318-25. doi:, 10.3109/10799890903295150. PMID:19903011 doi:http://dx.doi.org/10.3109/10799890903295150
  9. ↑ Weston C, Lu J, Li N, Barkan K, Richards GO, Roberts DJ, Skerry TM, Poyner D, Pardamwar M, Reynolds CA, Dowell SJ, Willars GB, Ladds G. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2). J Biol Chem. 2015 Sep 18;290(38):23009-22. doi: 10.1074/jbc.M114.624601. Epub, 2015 Jul 21. PMID:26198634 doi:http://dx.doi.org/10.1074/jbc.M114.624601
  10. ↑ Zhang X, Stevens RC, Xu F. The importance of ligands for G protein-coupled receptor stability. Trends Biochem Sci. 2015 Feb;40(2):79-87. doi: 10.1016/j.tibs.2014.12.005. Epub, 2015 Jan 15. PMID:25601764 doi:http://dx.doi.org/10.1016/j.tibs.2014.12.005
  11. ↑ 'Lehninger A., Nelson D.N, & Cox M.M. (2008) Lehninger Principles of Biochemistry. W. H. Freeman, fifth edition.'
  12. ↑ 12.0 12.1 Salon JA, Lodowski DT, Palczewski K. The significance of G protein-coupled receptor crystallography for drug discovery. Pharmacol Rev. 2011 Dec;63(4):901-37. doi: 10.1124/pr.110.003350. PMID:21969326 doi:http://dx.doi.org/10.1124/pr.110.003350

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