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

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] that induces insulin secretion.<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>. hGPR40 is highly expressed in human pancreatic [https://en.wikipedia.org/wiki/Beta_cell β cells], brain, and endocrine cells of the [https://en.wikipedia.org/wiki/Gastrointestinal_tract gastrointestinal tract].<ref name = "RenXM">PMID:26974599</ref> hGPR40 is of particular interest because the triggering of insulin secrection is [https://en.wikipedia.org/wiki/Glucose glucose] dependent.This glucose-dependence for hGPR40 signaling makes it a target for the treatment of [https://en.wikipedia.org/wiki/Diabetes_mellitus_type_2 type-2 diabetes] as agonists could increase glycemic control and lower the risk of hypoglycemia.<ref name="Srivastava"/> 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.

GPR40 is most prevalent in [https://en.wikipedia.org/wiki/Beta_cell pancreatic β-cells] where free fatty acids (FFAs) have pleiotropic effects <ref name="FFA">PMID:19460454</ref>. While acute intake of FFAs stimulates insulin release, chronic exposure to high levels of FFAs results in the impairment of β-cell function and insulin secretory response <ref name= "FFA"/>. GPR40 mediates the effect of both acute and chronic levels of FFAs. FFAs amplify glucose-stimulated insulin secretion from pancreatic β-cells by activating GPR40. When GPR40 is inhibited, insulin secretion no longer increases in response to fatty acid stimulation <ref name= "FFA"/>. This decreased activity of GPR40 leads to a decreased risk of [https://en.wikipedia.org/wiki/Hyperinsulinemia hyperinsulinemia], [https://en.wikipedia.org/wiki/Fatty_liver fatty liver disease], [https://en.wikipedia.org/wiki/Hypertriglyceridemia hypertriglyceridemia], [https://en.wikipedia.org/wiki/Hyperglycemia hyperglycemia], and [https://en.wikipedia.org/wiki/Impaired_glucose_tolerance glucose tolerance] in obese patients <ref name= "FFA"/>. On the contrary, overexpression of GPR40 leads to impaired β-cell function, hyperinsulinemia, and diabetes <ref name= "FFA"/>. These results suggest that GPR40 plays an important role in the mechanism that links obesity and type 2 diabetes and thus is a popular drug target being studied.  

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 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.

While there is relatively low sequence identity between hGPR40 and peptide-binding and [https://en.wikipedia.org/wiki/Opioid_receptor opioid GPCRs], they do share structural similarities such as a conserved <scene name='72/727085/Hairpin_loop/4'>hairpin loop</scene> motif on <scene name='72/727085/Ecl2/4'>extracellular loop 2 </scene>(ECL2).<ref name="Srivastava"/> In addition, there is a conserved <scene name='72/727085/Disulfide/3'>disulphide bond</scene> that is formed between transmembrane helix 3 (Cys 79) and the C-terminus of ECL2 (Cys170).<ref name="Srivastava"/> Compared to peptide-binding and opioid GPCRs which have distinctive [https://en.wikipedia.org/wiki/Beta_sheet β-sheets] spanning from transmembrane helix 4 to 5, hGPR40 possesses a shorter B-sheet-like region which has  [http://proteopedia.org/wiki/index.php/Image:Beta-like_factors_of_hGPR40_ECL2.png low B-factors].<ref name="Srivastava"/> This reflects the low mobility of the region that limits the overall flexibility of the adjacent portion of ECL2 between Leu171 and Asp175.<ref name="Srivastava"/> A unique feature of hGPR40 is the presence of an additional 13 residues (Pro147 to Gly159) on ECL2 which is absent on all the other peptide/opioid receptors.<ref name="Srivastava"/> These extra residues form a separate <scene name='72/727085/Auxiliary_loop/3'>auxiliary loop</scene> between the B-sheet-like region and transmembrane 4. Together, the auxiliary loop and ECL2 of hGPR40 function as a <scene name='72/727085/Ecl2_cap/3'>roof </scene> over the canonical binding site covering it from the central extracellular region.<ref name="Srivastava"/>

The canonical binding pocket for many other GPCRs is solvent exposed and centrally located between the transmembrane helices allowing ligands to directly bind from the extracellular space.<ref name="Srivastava"/> However, because the ECL2 acts as a roof to this canonical binding site, it inhibits ligands from entering directly from the extracellular region. Instead, the highly lipophilic nature of hGPRC40’s ligands allow it to enter a <scene name='72/727085/Hgpr40_entry/2'>noncanonical binding pocket </scene> between TM3 and TM4 by moving through the lipid bilayer.<ref name="Srivastava"/>  

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. 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"/>  

hGPR40 contains a highly conserved hairpin extracellular loop. This extracellular loop (<scene name='72/721541/Ecl2/4'>ECL2</scene>) is the longest and most divergent of the extracellular loops found in proteins (<scene name='72/721541/Ecl2_top/2'>top view of ECL2</scene>). The loop is accompanied by a [https://en.wikibooks.org/wiki/Structural_Biochemistry/Chemical_Bonding/_Disulfide_bonds disulfide bond] (<scene name='72/721541/Cysteine_bridge/3'>Cys79 and Cys170</scene>) that forms between transmembrane helix 4 and the C-terminus of the ECL2 loop. In hGPR40, ECL2 has two sections: a <FONT COLOR="#00FFFF">'''beta sheet'''</FONT> and an <FONT COLOR="#FF00FF">'''auxiliary loop'''</FONT>. The [https://en.wikipedia.org/wiki/Beta_sheet beta sheet] spans helices 4 and 5 and is shorter in hGPR40 than in other GPCRs. The ECL2 of hGPR40 also differs from that of other proteins because it contains an auxiliary loop of 13 extra residues. The entire extracellular loop has low mobility and flexibility which allows it to act as a cap for the binding pocket. The only exception to the low flexibility is the tip of the auxiliary loop, which corresponds to residues Asp152-Asn155. This area of greater mobility allows for substrates to enter the binding site.<ref name="Srivastava"/>

TAK-875 had the most promising outlooks out of any current known agonists of hGPR40, but it was discontinued. Some other agonists tested in clinical trials include AMG-837 and AM-1638. When coadministered, AMG-837 and AM-1638 enhanced glucose tolerance, but they were found to be toxic in the human trials. Some other agonsits are currently being examined as well. One compound, LY 2881835 (Eli Lilly & Company, Indianapolis, IN), has undergone clinical trials, but the results are unknown. In addition to the above-mentioned compound, other orally bioavailable GPR40-specific agonists are currently in preclinical or clinical  development. As of 2015, TUG-770 and CNX-011-67 (Connexios Life Sciences, Karnataka, India) were in preclinical trials and JTT-851 (Japan Tobacco, Toyko, Japan), and P11187 (Piramal, Mumbai, India) were in clinical trails.<ref name="Mancini">PMID: 25604916</ref>