User:R. Jeremy Johnson/GPR40: Difference between revisions

<StructureSection load='Sele4phu.pdb' size='400' side='right' caption='Human G-Protein Receptor 40 (hGPR40) visualized at 2.3 Å resolution by X-ray crystallography (PDB: [http://www.rcsb.org/pdb/explore/explore.do?structureId=4phu 4PHU]). The natural substrates of this protein are free fatty acids, giving rise to its secondary name, Free Fatty Acid Receptor 1 (FFAR1).' scene='72/726431/General_protein/1'>

Human G-protein coupled receptor 40 (hGPR40), also known as free fatty acid 1 receptor (FFAR1), is a seven helical transmembrane domain receptor for long-chain free [https://en.wikipedia.org/wiki/Fatty_acid fatty acids].<ref name="Srivastava">PMID:25043059</ref> Some known fatty acid substrates of hGPR40 include [http://www.news-medical.net/health/What-is-Linoleic-Acid.aspx linoleic acid], [http://www.livestrong.com/article/438717-what-is-oleic-acid/ oleic acid], [http://www.hmdb.ca/metabolites/hmdb02925 eicosatrienoic acid], and [https://en.wikipedia.org/wiki/Palmitoleic_acid palmitoleic acid]<ref name="Morgan">PMID:19660440</ref>. This protein is primarily located in the [https://en.wikipedia.org/wiki/Beta_cell pancreatic β-cells] in the [https://en.wikipedia.org/wiki/Pancreatic_islets islets of Langerhans], and as such, it has become a target for potential [http://www.diabetes.org/living-with-diabetes/treatment-and-care/ Type 2 Diabetes treatments] because hGPR40 stimulates insulin secretion.<ref name="Kebede">PMID:22308370</ref> Type 2 diabetes is especially relevant because the cells have become desensitized to insulin and free fatty acids. Activation of hGPR40 stimulates insulin secretion and decreases extracellular glucose concentration.<ref name="Ma">PMID:26620255</ref> GPR40 is a member of a group of homologous [[GPCRs]] all located on chromosome 19q13.1 including GPCR41, 42, and 43.<ref name="Burant">PMID:23882043</ref> Evidence exists that shows GPCR43 is involved in adipogenesis. GPCR41 was also previously believed to participate in [http://jcs.biologists.org/content/124/16/2681 adipogenesis], but this was shown to be false.<ref name="Hong">PMID:16123168</ref>

Like most G-protein coupled receptors, hGPR40 contains <scene name='72/721541/Top_view_transmembrane_helices/2'>seven transmembrane helices</scene> (<scene name='72/721541/Top_view_transmembrane_helices/1'>top view of TM helices</scene>). To obtain a [https://en.wikipedia.org/wiki/Protein_crystallization crystallized structure] of the protein, four <scene name='72/721541/Stabilizing_mutations/4'>stabilizing mutations</scene> (<scene name='72/721541/L42a/3'>L42A</scene>, <scene name='72/721541/F88a/4'>F88A</scene>, <scene name='72/721541/G103a/3'>G103A</scene>, <scene name='72/721541/Y202f/3'>Y202F</scene>) were made to increase expression levels and thermal stability of the protein. These mutations did not significantly impact the enzyme's binding affinity with a known agonist, TAK-875.<ref name="Srivastava"/> A <scene name='72/721541/Lysozyme_crimson/2'>T4 Lysozyme</scene> (shown in <FONT COLOR="#DC143C">'''crimson'''</FONT>) was also added to intracellular loop 3 to aid in the formation of crystals. T4 Lysozyme also had little effect on TAK-875 binding.<ref name="Srivastava"/> For clarity, lysozyme is removed in all further renderings of hGPR40. hGPR40 also contains an extracellular loop that is conserved among most G-protein coupled receptors (ECL2). This loop has two subsections and is involved in the permeability of the binding site.

[[Image:HGPR40bind3.png|200 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). The location of this binding site with respect to the membrane proposes that substrates would gain entry to the membrane by binding in this site. Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins that exhibit cooperativity are regulated by the binding of inhibitors.

[[Image:hydrogen bonding black.png|200 px|right|thumb|Figure 3. TAK-875 with key binding residues Tyr91, Arg183, Tyr240, and Arg258. These residues all hydrogen bond to the carboxylate moiety of TAK-875.]]hGPR40 has a distinct binding pocket that is established by <scene name='72/721541/All_binding_residues/3'>eight key residues</scene>: <scene name='72/721541/Tyr91/1'>Tyr91</scene>, <scene name='72/721541/Glu172/2'>Glu172</scene>, <scene name='72/721541/Arg183/2'>Arg183</scene>, <scene name='72/721541/Ser187/2'>Ser187</scene>, <scene name='72/721541/Tyr240/1'>Tyr240</scene>, <scene name='72/721541/Asn241/1'>Asn241</scene>, <scene name='72/721541/Asn244/1'>Asn244</scene>, and <scene name='72/721541/Arg258/1'>Arg258</scene> (all individual residues shown in <FONT COLOR="#00FF00">'''chartreuse'''</FONT>). The importance of these residues for agonist binding was determined by alanine [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis mutagenesis] studies. Each of these residues have either a [http://www.proteinstructures.com/Structure/Structure/amino-acids.html charged or polar R-group] that creates a charge network that keeps these residues in a stable, unbound state until exposed to a substrate. When the substrate (an agonist) enters the binding pocket, four of the eight <scene name='72/721541/Hydrogen_binding_1/8'>key binding residues</scene> interact directly with the carboxylate moiety of the agonist by hydrogen bonding to it. These residues include two key arginines in the binding pocket, Arg183 and Arg258,<ref name="Sum">PMID: 17699519</ref><ref name="Sum, C.">PMID:19068482</ref> and two key tyrosine residues, Tyr91 and Tyr240 (Figure 3). Tyr240 is especially important for binding, as mutation of Tyr240 caused an eight fold reduction in the binding affinity of TAK-875 and had a significant effect on the [https://en.wikipedia.org/wiki/Dissociation_constant K_D] of the protein.<ref name="Srivastava"/>  

One proposed pathway of insulin secretion by hGPR40 involves the activation of the [https://en.wikipedia.org/wiki/Gq_alpha_subunit G_aq/11] protein complex. This complex then activates [[phospholipase C]] (PLC) which in turn hydrolyses [https://en.wikipedia.org/wiki/Phosphatidylinositol_4,5-bisphosphate phosphatidylinositol 4,5-bisphosphate] to inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ can then mediate the [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560308/ influx of Ca²⁺] by moving into the cytoplasm, binding to the endoplasmic reticulum, and allowing for the release of Ca²⁺ into the cytosol.<ref name="Burant"/> This increase in [Ca²⁺] amplifies the similar increase in [Ca²⁺] that results from high concentrations of glucose. In this way, hGPR40 mimics glucose dependent insulin secretion.<ref name="Itoh">PMID:12629551</ref> Overall, hGPR40 helps to amplify the Ca²⁺ signal so that the cell secretes more insulin.

Another pathway through which hGPR40 may induce insulin expression is through phospholipase D1 (PKD1). When free fatty acids bind to hGPR40, hGPR40 directly phosphorylates and activates PKD1. The PKD1 plays a role in controlling the organization of an actin network that plays in role in insulin secretion.<ref name="Burant"/>

[[Image:Simplified hydrophibic.png|380 px|center|thumb|'''Figure 4:''' Stabilizing binding interactions between TAK-875 and hGPR40. Amino acids denoted in orange coordinate with the polar head of TAK-875 by polar/ionic interactions while blue amino acids stabilize the binding through hydrophobic interactions.]]

== Signal Transduction ==

 

[[Image:Gpr40 insulin pathway.png|400 px|center|thumb|'''Figure 5:''' Signaling pathway mediated by GPR40 in pancreatic β-cells. The 𝝰q subunit represents the Gq unit that is coupled to hGPR40. This subunit breaks off to activate phospholipase C (PLC), resulting in the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP₂). PIP₂ goes on to create diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The DAG activates protein kinase c (PKC), triggering secretion of insulin. Activated IP3 diffuses to the endoplasmic reticulum (ER), releasing the stored Ca²⁺, which aids in insulin secretion. ]]

 

The natural substrate of hGPR40 is free fatty acids (FFAs) which bind to the G-protein and enhance glucose-stimulated insulin secretion <ref name="Morg">PMID:19660440</ref>. FFAs bind to GPR40 which then couples with the G-protein Gq leading to increased [https://en.wikipedia.org/wiki/Phospholipase_C phospholipase C] (PLC) activity<ref name="Morg"/>. PLC catalyzes the hydrolysis of the phospholipid [https://en.wikipedia.org/wiki/Phosphatidylinositol_4,5-bisphosphate phosphatidylinositol-4,5-biphosphate ] (PIP₂) resulting in the formation of [https://en.wikipedia.org/wiki/Diglyceride diacylglycerol] (DAG) and [https://en.wikipedia.org/wiki/Inositol_trisphosphate inositol 1,4,5-triphosphate] (IP3)<ref name="Morg"/>. DAG can then activate [https://en.wikipedia.org/wiki/Protein_kinase_C protein kinase C] (PKC) to enhance insulin secretion<ref name="Morg"/>. IP3 on the other hand is soluble and diffuses to the [https://en.wikipedia.org/wiki/Endoplasmic_reticulum endoplasmic reticulum] where it binds a ligand-gated Ca²⁺ channel<ref name="Morg"/>. This binding triggers the opening of the channel causing stored Ca²⁺ to be released into the cytoplasm<ref name="Morg"/>. This large increase in intracellular free Ca²⁺ produces a proportional increase in glucose-dependent insulin secretion, suggesting that insulin release can be contributed in part to the changes in Ca²⁺ concentration resulting from activated GPR40 <ref name="Morg"/>.