Name: quantifying agonist activity at g protein-coupled receptors
Uploaded: 2011-12-26T12:00:00
Description: Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Dette arbejde blev støttet af en National Institutes of Health Grant GM-69829.

1. Måling af agonist koncentrations-respons kurver: ingen konstitutiv aktivitet <ol><li> Til estimering af relativ agonist K b værdier (RA i), er en serie på mindst to agonist koncentrations-respons kurver påkrævet. Enhver in vitro analyse til den funktionelle respons af en GPCR kan måles forudsat, at koncentrationen af agonist kan kontrolleres, og en enkelt type receptor formidler respons. Egnet cellebaserede assays omfatter måling af cAMP 4,7 og inos...

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Fordi vores metode til at anslå RA i (relativ K b værdi) kræver kun måling af agonist koncentration-respons kurver, kan vores analyse gøres når som helst disse kurver måles. Hvis dag-til-dag variation i svaret fra den eksperimentelle forberedelse (f.eks celler eller væv) er stor, kan svaret målinger af hver koncentration-responskurve være normaliseret i forhold til &quot;Top&quot; estimat af standarden agonist for hver dag, eksperiment. Et sk...

quantifying agonist activity at g protein-coupled receptors

Når en agonist aktiverer en population af G-protein-koblede receptorer (GPCRs), det fremkalder en signalvej, der kulminerer i svaret fra den celle eller væv. Denne proces kan analyseres på niveau med en enkelt receptor, en befolkning på receptorer, eller en downstream svar. Her beskriver vi, hvordan at analysere det efterfølgende reaktion at opnå et estimat af de agonist affinitetskonstant til aktiv tilstand enkelt receptorer. Receptorer opfører sig som kvantemekaniske switche, der veksler mellem aktive og inaktive stater (Figur 1). Den aktive tilstand interagerer med specifikke G proteiner eller andre signalering partnere. I mangel af ligander, dominerer den inaktive tilstand. Bindingen af agonist øges sandsynligheden for, at receptoren vil skifte til aktiv tilstand, fordi dens affinitetskonstant til aktiv tilstand (K b) er meget større end for den inaktive tilstand (K a). Sammenlægning af detilfældige udgange af alle de receptorer i befolkningen giver et konstant niveau af receptor-aktivering i tide. Den reciprokke værdi af koncentrationen af agonist fremkalde halv maksimal receptor aktivering svarer til den observerede affinitetskonstant (K OBS), og den fraktion af agonist-receptor komplekser i den aktive tilstand er defineret som effekten (ε) (Figur 2). Metoder til at analysere de efterfølgende reaktioner GPCRs er blevet udviklet, der muliggør estimering af K OBS og relative effekt af en agonist 1,2. I denne rapport, viser vi, hvordan du ændrer denne analyse at estimere agonist K b værdi i forhold til en anden agonist. For analyser som udviser konstitutive aktivitet, viser vi, hvordan at estimere K b i absolutte enheder af M -1. Vores metode til at analysere agonist koncentrations-respons kurver 3,4 beståraf den globale ikke-lineær regression ved hjælp af operationelle model 5. Vi beskriver en procedure, ved hjælp af programmet, Prism (GraphPad Software, Inc., San Diego, CA). Analysen giver et estimat af produktet af K obs. og en parameter i forhold til effekten (τ). Skønnet over τK obs. af en agonist, divideret med en anden, er et relativt mål for K b (RA i) 6. For enhver receptor udstille konstitutiv aktivitet, er det muligt at estimere et parameter i forhold til effekten af det frie receptor komplekset (τ sys). I dette tilfælde er K b værdien af en agonist, der svarer til τK obs. / τ sys 3. Vores metode er nyttig til bestemmelse af selektivitet af en agonist for receptor subtyper og for kvantificering af agonist-receptor-signalering gennem forskellige G-proteiner.

Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Quantifying Agonist Activity at G Protein-coupled Receptors

En metode til at anslå affinitetskonstant af en agonist for den aktive tilstand (<em&gt; K<sub&gt; B</sub</em&gt;) Af en G-protein-koblede receptorer er beskrevet. Analysen giver absolut eller relativ foranstaltninger<em&gt; K<sub&gt; B</sub</em&gt; Afhængig af, om konstituerende receptor-aktivering er målbar. Vores metode gælder for de forskellige svar nedstrøms fra receptor aktivering.

Kvantificering agonistaktivitet på G-protein-koblede receptorer

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

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