जी प्रोटीन युग्मित रिसेप्टर्स पर agonist गतिविधि को बढ़ाता
Summary
आत्मीयता सक्रिय राज्य के लिए एक agonist के निरंतर आकलन के लिए एक विधि (<em> कश्मीर<sub> ख</sub</em> जी रिसेप्टर प्रोटीन युग्मित) में वर्णित है. विश्लेषण के निरपेक्ष या रिश्तेदार उपाय प्रदान करता है<em> कश्मीर<sub> ख</sub</em> पर निर्भर करता है कि विधान रिसेप्टर सक्रियण औसत दर्जे का है. हमारी विधि रिसेप्टर सक्रियण से विभिन्न प्रतिक्रियाएं बहाव के लिए लागू होता है.
Abstract
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 or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (τ). The estimate of τKobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τsys). In this case, the Kb value of an agonist is equivalent to τKobs/τsys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.
Protocol
Discussion
क्योंकि आरए मैं (सापेक्ष कश्मीर ख मूल्य) का आकलन करने के लिए हमारे विधि केवल एकाग्रता प्रतिक्रिया agonist घटता के माप की आवश्यकता है, हमारे विश्लेषण के किसी भी समय इन घटता मापा जाता है किया जा सकत…
Declarações
The authors have nothing to disclose.
Acknowledgements
यह काम स्वास्थ्य अनुदान जीएम 69,829 के एक राष्ट्रीय संस्थान द्वारा समर्थित किया गया.
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