Functional G-Protein-Coupled Receptor (GPCR) Activation Assay (35SGTPγS Binding)
The 35SGTPγS assay allows for the assessment of functional activation of Gi/Go-coupled receptors by measuring bound 35SGTPγS in brain sections using autoradiography. This assay is useful for investigating biased agonism in μ-opioid receptors (MORs).
MOR agonist DAMGO stimulates 35SGTPγS binding in the rat spinal cord, amygdala, and striatum/accumbens. Pre-incubation with the opioid receptor antagonist, naloxone, blocks DAMGO-stimulated 35SGTPγS binding. Slides were loaded into the cassette of a Beta-imager 2000 (BioSpace Lab, France), and the images of the brain sections were acquired for 20 hours. Surface activity (cpm/mm2) was measured for specific regions using Beta vision + software (BioSpace Lab, France). The percent increase of DAMGO-stimulated binding over basal binding was calculated as: [DAMGO (cpm/mm2)-basal (cpm/mm2)]/basal (cpm/mm2) X 100%] and was about 104% for spinal cord, 89% for striatum/accumbens, and 72% for amygdala.
