ABSTRACT
BACKGROUND: G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization. OBJECTIVE: To develop a quantitative fluorescence Microscopy assay to study ß2- Adrenergic receptor expression and desensitization. METHOD: ß2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of ß2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software. RESULTS: The method is sensitive and allows for the simultaneous measurement of surface and total expression of ß2-AR. CONCLUSION: A highly accurate method is described for measuring ß2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of ß2-Adrenergic receptor and can be applied to essentially any other GPCR.
Subject(s)
Receptors, Adrenergic, beta-2/metabolism , Animals , Humans , Microscopy, Fluorescence , Receptors, Adrenergic, beta-2/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Single-Cell Analysis , SoftwareABSTRACT
The molecular mechanisms by which individuals subjected to environmental heat stress either recover or develop heat-related complications are not well understood. We analysed the changes in blood mononuclear gene expression patterns in human volunteers exposed to extreme heat in a sauna (temperature of 75.7 ± 0.86 °C). Our analysis reveals that expression changes occur rapidly with no significant increase in core temperature and continue to amplify one hour after the end of heat stress. The reprogramed transcriptome was predominantly inhibitory, as more than two-thirds of the expressed genes were suppressed. The differentially expressed genes encoded proteins that function in stress-associated pathways; including proteostasis, energy metabolism, cell growth and proliferation, and cell death, and survival. The transcriptome also included mitochondrial dysfunction, altered protein synthesis, and reduced expression of genes -related to immune function. The findings reveal the human transcriptomic response to heat and highlight changes that might underlie the health outcomes observed during heat waves.
