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Contents 1 Introduction 2 Structural Considerations 2.1 Comparison between Class A and Class B GPCRs 2.2 Structurally Significant GCGR 7TDM Residues 3 Functions of Glucagon receptor (GCGR) 3.1 Peptide binding and selectivity 3.2 Conformational changes 3.3 Signaling pathways 4 Kinetics 5 The Signal Peptide: Glucagon 5.1 Biological function of glucagon 5.2 Glucagon Peptide Stability and Active Binding Sites 5.3 Active binding domains/sites 6 Clinical relevance 6.1 Future research direction 6.2 Current drug targets 6.3 Possible structural considerations for agonists

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)

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] [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]

Structurally Significant GCGR 7TDM Residues

The snake plot 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 of the TMD providing secondary recognition. ^[6]

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, but research is ongoing. ^[8]

Signaling pathways

GCGR signals generate 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 regulates 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 the 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. This allows the body to export the fatty acid to the liver and other crucial tissues for energy use and makes more glucose available for use in brain functioning. Through these processes, glucagon is able to increase glucose levels in the blood. Glucagon lowers the concentration of fructose 2,6-bisphosphate in the blood, which is an allosteric inhibitor of gluceogenesis. Glucagon also promotes the functioning of the 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

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

See “Landmark studies on the glucagon subfamily of GPCRs: from small molecule modulators to a crystal structure” good clinical references and small molecule target tables

Possible structural considerations for agonists

(our distance measurements and shape analysis images) Also see Landmark studies on the glucagon subfamily of GPCRs: from small molecule modulators to a crystal structure

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