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Development of sensitive cellular assay systems and their application to the identification of "orphan" seven-transmembrane receptors

Kotarsky, Knut LU (2003)
Abstract (Swedish)
Popular Abstract in Swedish

I alla flercelliga organismer behövs utbyte av information mellan cellerna, som bygger upp kroppen. För detta ändamål är cellerna försedda med små antenner, som kan ta emot signaler och kallas därför receptorer. Receptorer kan vara uppbyggda på olika sätt. De flesta receptorer slingrar sig sju gånger genom membranet och har ett gemensamt sätt att fortleda signaler till insidan av cellen via så kallade G-proteiner. De kallas därför 7TM (transmembran) eller G-protein kopplade receptorer. Dessa receptorer är mycket betydelsefula då de styr många fysiologiska funktioner i kroppen, men även eftersom de utgör måltavlor för många läkemedel. De är faktiskt de mål på cellytan, som flest läkemedel använder... (More)
Popular Abstract in Swedish

I alla flercelliga organismer behövs utbyte av information mellan cellerna, som bygger upp kroppen. För detta ändamål är cellerna försedda med små antenner, som kan ta emot signaler och kallas därför receptorer. Receptorer kan vara uppbyggda på olika sätt. De flesta receptorer slingrar sig sju gånger genom membranet och har ett gemensamt sätt att fortleda signaler till insidan av cellen via så kallade G-proteiner. De kallas därför 7TM (transmembran) eller G-protein kopplade receptorer. Dessa receptorer är mycket betydelsefula då de styr många fysiologiska funktioner i kroppen, men även eftersom de utgör måltavlor för många läkemedel. De är faktiskt de mål på cellytan, som flest läkemedel använder sig av. I detta arbete beskrivs nya förfinade metoder att mäta signalfortledningen genom sådana receptorer (delarbeten I och II). Det kan användas för att identifiera substanser (läkemedel eller naturliga ämnen) som kan binda till, och aktivera receptorer. Utöver detta beskrivs identifieringen av två nya receptorer (delarbeten III och IV), som ingår i superfamiljen. Den första receptorn blev inledningsvis identifierad från databasen för det humana genomet, som ett potentiell receptor med stor likhet med den redan kända leukotrien-receptorn, BLT1. Vidare utnyttjades receptorn för att hitta den substans, som binder och aktiverar receptorn i kroppen. Substansen visade sig vara leukotriene B4, en signalsubstans som frisätts när till exempel infektioner pågår och som rekryterar immunceller. Fysiologiska funktionen för den andra leukotriene B4 receptorn, BLT2 är ännu inte helt klarlagd. Den andra receptorn är den första receptorn som aktiveras av fettsyror. Receptorn finns i många organ i kroppen som är förknippade med energiomsättningen och metabolism, såsom levern, muskelvävnad och hjärta. Men receptorn uttrycks också i de insulin-producerande celler, som finns i bukspottkörteln. Receptorn är inblandad i den insulin-frisättning, som äger rum efter en fettrik måltid. Vad som gör receptorn extra betydelseful är, att även nya läkemedel mot diabetes typ II interagerar med receptorn. Det är möjligt att delar av läkemedelsinverkan överförs genom den nya receptor. Upptäckten kan på så vis påverka utvecklingen av nya och bättre antidiabetes-läkemedel och kanske förkorta utvecklingstiden. (Less)
Abstract
Seven-transmembrane, G-protein coupled receptors play a central role in physiology since they facilitate cell communication in multicellular organisms by recognition of a broad range of ligands. They also represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and, hence, their physiological functions, remain to be identified. These receptors are usually referred to as “orphan” receptors. A pre-requisite for the identification of ligands activating “orphan” receptors is powerful assay systems displaying a high assay quality as specified by a high Z-value. Until now, reporter gene assays have not been in common use in this process. Therefore, we aimed to develop improved reporter gene assays.... (More)
Seven-transmembrane, G-protein coupled receptors play a central role in physiology since they facilitate cell communication in multicellular organisms by recognition of a broad range of ligands. They also represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and, hence, their physiological functions, remain to be identified. These receptors are usually referred to as “orphan” receptors. A pre-requisite for the identification of ligands activating “orphan” receptors is powerful assay systems displaying a high assay quality as specified by a high Z-value. Until now, reporter gene assays have not been in common use in this process. Therefore, we aimed to develop improved reporter gene assays. This aim was accomplished by optimizing the promoter region of the construct, the reporter enzyme, and the assay procedure. Furthermore, a fluorescence-based clone selection step was introduced, that allowed the selection of the most sensitive reporter cell clones. The established test cell lines responded sensitively upon stimulation of various cell surface receptors as demonstrated by several receptors. In the first approach, transcription of the reporter gene was under control of a synthetic promoter consisting of 9 TPA responsive elements. In a further improved construct, the promoter was extended with six NF-kB and six STAT motifs. The used reporter gene was designed as a fusion gene coding for green fluorescent protein and Photinus luciferase. The amplification of reporter enzyme activity was substantially larger than in any other described system, and the high assay quality made it suitable as a primary screening tool. The development of efficient assays allowed the screening for hitherto unknown ligands to orphan seven-transmembrane receptors. Natural ligands for two recently unknown receptors were identified. Thus, we identified the second leukotriene B4 receptor, BLT2, and the first cell surface, free fatty acid receptor, FFA1. The BLT2 receptor was first identified in silico, cloned, and subsequently functionally expressed in HeLa cells. The identification of FFA1 was accomplished using a reverse pharmacology approach. The FFA1 receptor (previously known as GPR40) responded to medium to long chain free fatty acids, including compounds like ±9-hydroxy-octadecadienoic acid (9-HODE) and a conjugated linoleic acid (10,12-CLA). Receptor expression was detected in heart, skeletal muscle, liver and in pancreatic b-cells. Most importantly, the identification of anti-diabetic drugs (thiazolidinediones and MEDICA16) as agents acting on this receptor implies an important connection between FFA1R and type II diabetes. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Svensson, Samuel, Associate Professor of Pharmacology; Linköping University; Associate Principle Scientist; AstraZeneca Södertälje
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biokemi, Metabolism, Biochemistry, metabolism, leukotrieneB4, thiazolidinedione, 9-HODE, fatty acid, luciferase, GFP, reporter gene, preclinical assay, receptor, GPCR
pages
96 pages
publisher
Knut Kotarsky; Tordmulevägen 3c; 22735 LUND Sweden,
defense location
Segerfalksalen; Sölvegatan 17 LUND
defense date
2003-05-03 10:15
external identifiers
  • Other:ISRN: LUMED/MEFN-34-SE
ISBN
91-628-5595-6
language
English
LU publication?
yes
id
a6c17df1-b05b-4de7-a491-19cabb40136f (old id 465640)
date added to LUP
2007-09-27 10:44:37
date last changed
2016-09-19 08:45:07
@misc{a6c17df1-b05b-4de7-a491-19cabb40136f,
  abstract     = {Seven-transmembrane, G-protein coupled receptors play a central role in physiology since they facilitate cell communication in multicellular organisms by recognition of a broad range of ligands. They also represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and, hence, their physiological functions, remain to be identified. These receptors are usually referred to as “orphan” receptors. A pre-requisite for the identification of ligands activating “orphan” receptors is powerful assay systems displaying a high assay quality as specified by a high Z-value. Until now, reporter gene assays have not been in common use in this process. Therefore, we aimed to develop improved reporter gene assays. This aim was accomplished by optimizing the promoter region of the construct, the reporter enzyme, and the assay procedure. Furthermore, a fluorescence-based clone selection step was introduced, that allowed the selection of the most sensitive reporter cell clones. The established test cell lines responded sensitively upon stimulation of various cell surface receptors as demonstrated by several receptors. In the first approach, transcription of the reporter gene was under control of a synthetic promoter consisting of 9 TPA responsive elements. In a further improved construct, the promoter was extended with six NF-kB and six STAT motifs. The used reporter gene was designed as a fusion gene coding for green fluorescent protein and Photinus luciferase. The amplification of reporter enzyme activity was substantially larger than in any other described system, and the high assay quality made it suitable as a primary screening tool. The development of efficient assays allowed the screening for hitherto unknown ligands to orphan seven-transmembrane receptors. Natural ligands for two recently unknown receptors were identified. Thus, we identified the second leukotriene B4 receptor, BLT2, and the first cell surface, free fatty acid receptor, FFA1. The BLT2 receptor was first identified in silico, cloned, and subsequently functionally expressed in HeLa cells. The identification of FFA1 was accomplished using a reverse pharmacology approach. The FFA1 receptor (previously known as GPR40) responded to medium to long chain free fatty acids, including compounds like ±9-hydroxy-octadecadienoic acid (9-HODE) and a conjugated linoleic acid (10,12-CLA). Receptor expression was detected in heart, skeletal muscle, liver and in pancreatic b-cells. Most importantly, the identification of anti-diabetic drugs (thiazolidinediones and MEDICA16) as agents acting on this receptor implies an important connection between FFA1R and type II diabetes.},
  author       = {Kotarsky, Knut},
  isbn         = {91-628-5595-6},
  keyword      = {Biokemi,Metabolism,Biochemistry,metabolism,leukotrieneB4,thiazolidinedione,9-HODE,fatty acid,luciferase,GFP,reporter gene,preclinical assay,receptor,GPCR},
  language     = {eng},
  pages        = {96},
  publisher    = {ARRAY(0x8bc4ea8)},
  title        = {Development of sensitive cellular assay systems and their application to the identification of "orphan" seven-transmembrane receptors},
  year         = {2003},
}