10.3791/54088-v

Biomass Conversion to Produce Hydrocarbon Liquid Fuel Via Hot-vapor Filtered Fast Pyrolysis and Catalytic Hydrotreating

10.3791/54111-v 09:04 min

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

10.3791/54144-v 08:27 min

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

10.3791/54286-v C

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

10.3791/54287-v 12:00 min

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

10.3791/54391-v 10:53 min

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

10.3791/54396-v 12:24 min

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

10.3791/54409-v 08:01 min

Preparation of Homogeneous MALDI Samples for Quantitative Applications

10.3791/54420-v

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

10.3791/54458-v 10:21 min

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

10.3791/54463-v

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

10.3791/54466-v

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

作者： Chirag Vasavda*1, Nicholas W. Zaccor*1, Paul C. Scherer1, Charlotte J. Sumner1,2, Solomon H. Snyder1,3,4 1The Solomon H. Snyder Department of Neuroscience,Johns Hopkins University School of Medicine, 2Department of Neurology and neurosurgery,Johns Hopkins University School of Medicine, 3Departments of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, 4Department of Psychiatry and Behavioral Sciences,Johns Hopkins University School of Medicine

简介：

Overview

This protocol outlines a method to pharmacologically characterize G-protein-coupled receptor (GPCR) interactions by monitoring the binding of a radio-labeled GTP analog. It provides insights into the pharmacologic properties of GPCRs, which are crucial for understanding cellular signaling.

Key Study Components

Area of Science

Biochemistry
Pharmacology
Cell Biology

Background

GTP binding is an early event in GPCR activation.
Understanding GPCR pharmacology is essential for drug development.
This method allows for the study of both established and orphan GPCRs.
It is a simple and rapid assay for measuring GPCR signaling.

Purpose of Study

To isolate cellular membranes containing GPCRs.
To study the pharmacologic properties of GPCR-ligand interactions.
To provide a functional assay for GTP binding.

Methods Used

Plate human embryonic kidney cells in tissue culture plates.
Incubate cells until confluency is reached.
Transfect cells with HAMOR1 using a transfection reagent.
Monitor GTP binding using [35S]GTPγS.

Main Results

Successful isolation of GPCR-containing membranes.
Characterization of GPCR-ligand interactions.
Measurement of GTP binding as a function of ligand presence.
Insights into the pharmacologic properties of GPCRs.

Conclusions

The method provides a reliable approach to study GPCR pharmacology.
It can be applied to both known and orphan GPCRs.
This assay contributes to the understanding of GPCR signaling pathways.

Frequently Asked Questions

What is the significance of GTP binding in GPCR activation?

GTP binding is one of the earliest events in the activation of GPCRs, initiating the signaling cascade.

How does this method contribute to drug development?

By characterizing GPCR-ligand interactions, this method aids in identifying potential drug targets.

What types of receptors can be studied using this protocol?

Both established and orphan GPCRs can be studied using this method.

What are the advantages of this GTP binding assay?

It is simple, rapid, and measures the most proximal event in GPCR signaling.

What cell type is used in this protocol?

Human embryonic kidney cells are used for the assay.

What is the role of [35S]GTPγS in this study?

[35S]GTPγS is a radio-labeled GTP analog used to monitor GTP binding in response to ligands.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [³⁵S]guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPγS), in response to a ligand of interest.