Characterizing a GPCR-ligand interaction is critical to understanding the biology of the receptor. As GDP/GTP exchange is one of the earliest events that follows ligand binding, monitoring GTP binding can measure GPCR activation or inhibition. Assaying more downstream events in GPCR signaling is often not as quantitative or stoichiometric, may not distinguish full agonists from partial ones, and can require expensive reagents. Moreover, increased GTP binding to Gα proteins is an almost-universal event following GPCR activation, meaning that measuring GTP binding is a broadly applicable assay for monitoring the activity of most GPCRs. Measuring GTP binding is a simple and rapid approach to monitor GPCR signaling in cells overexpressing the receptor of interest or in native tissue. The present protocol details a functional GTP-binding assay using an archetypal GPCR, the µ-opioid receptor (MOR1), to quantitatively determine the activity of an agonist and antagonist on GPCR signaling.
But we are facing some logistical issues acquiring the ([35S]GTPγS), and due to shortage of time we need to measure the GPCR signaling without the radio-labeled ([35S]GTPγS).
Could you please help me out and suggest some alternate approach?
I suggest using fluorescently labelled GTPgammaS like this:
https://www.jenabioscience.com/nucleotides-nucleosides/nucleotides-by-structure/non-hydrolyzable-nucleotides/gamma-phosphate-modified-nucleotides/guanosine-nucleotides/nu-209-mant-gtpgammas
This way you can follow fluorescence intensity changes in membrane preps but perhaps with significant background, so you should use GPCR overexpressing cell line for membrane prep. Sorry, I do not have protocol, so look for references. It should work.
There are several alternative methods available to measure G protein-coupled receptor (GPCR) signaling without relying on radio-labeled GTP binding. These methods provide valuable insights into GPCR activation and downstream signaling events. Here are some commonly used techniques:
1. Fluorescent GTP Analogs: Fluorescently labeled GTP analogs, such as mant-GTP or Bodipy-GTP, can be used to monitor GTP binding to G proteins. These analogs emit fluorescence upon binding to G proteins, allowing for real-time detection of GPCR activation. Fluorescence resonance energy transfer (FRET) techniques can also be employed to measure GTP binding indirectly.
2. Bioluminescence Resonance Energy Transfer (BRET): BRET is a proximity-based assay that measures protein-protein interactions. In the context of GPCR signaling, BRET can be used to detect interactions between GPCRs and downstream signaling molecules, such as G proteins or β-arrestins. This technique provides insights into GPCR activation and downstream signaling events.
3. Calcium Imaging: GPCR activation often leads to intracellular calcium mobilization. Calcium imaging techniques, such as fluorescence microscopy using calcium-sensitive dyes (e.g., Fluo-4), can be employed to measure changes in intracellular calcium levels upon GPCR activation. These measurements provide information about GPCR signaling and downstream calcium-dependent processes.
4. Phosphorylation Assays: GPCR activation often triggers phosphorylation events mediated by protein kinases. Phosphorylation assays, such as western blotting or immunofluorescence using phospho-specific antibodies, can be utilized to detect and quantify GPCR-induced phosphorylation of downstream signaling proteins. This approach provides insights into GPCR signaling pathway activation.
5. cAMP Assays: GPCRs can activate intracellular signaling pathways, such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP assays, such as enzyme-linked immunosorbent assays (ELISAs) or fluorescence-based assays using cAMP-sensitive dyes, can be employed to measure changes in cAMP levels upon GPCR activation. These measurements provide information about GPCR signaling and downstream cAMP-dependent processes.
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