Approaches to Characterize and Quantify Oligomerization of GPCRs
Sara Marsango1, María José Varela, Graeme Milligan
1Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ, Scotland, UK.
Methods in Molecular Biology (Clifton, N.J.)
|August 12, 2015
Summary
This study applies homogeneous time-resolved FRET (htrFRET) with Tag-lite™ technology to analyze protein interactions and GPCR homomer formation in live cells. The method efficiently quantifies these interactions within the plasma membrane.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Fluorescence resonance energy transfer (FRET) is a key technique for studying molecular proximity in live cells.
- Quantifying FRET signals is crucial for understanding biological processes like protein-protein interactions.
- Existing FRET methods have limitations in live-cell applications.
Purpose of the Study:
- To present a novel application of homogeneous time-resolved FRET (htrFRET) combined with Tag-lite™ technology.
- To demonstrate the utility of this method for detecting protein-protein interactions in real-time.
- To assess the homomerization capability of G protein-coupled receptor (GPCR) mutant variants compared to wild-type GPCRs.
Main Methods:
- Utilized homogeneous time-resolved FRET (htrFRET) for signal detection.
- Employed Tag-lite™ technology for enhanced FRET measurements.
- Applied the method to transfected cells to analyze protein interactions at the plasma membrane.
Main Results:
- Successfully quantified protein-protein interactions using htrFRET and Tag-lite™.
- Determined the homomerization status of GPCR mutant variants.
- Compared homomer formation between mutant and wild-type GPCRs within the cell membrane.
Conclusions:
- htrFRET combined with Tag-lite™ is a robust method for studying protein interactions in live cells.
- This approach effectively differentiates homomerization capabilities of GPCR variants.
- The technology provides valuable insights into GPCR function and molecular interactions within the plasma membrane.
More Related Videos
Related Concept Videos
01:15G Protein-coupled Receptors
19.8K
G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
19.8K
01:21G-protein Coupled Receptors
134.4K
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
134.4K
01:21G-protein Coupled Receptors
7.1K
7.1K
01:30Transducer Mechanism: G Protein–Coupled Receptors
8.2K
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical,...
GPCRs are also called heptahelical,...
8.2K
01:29The Two-State Receptor Model
3.5K
The two-state receptor model explains a drug's interaction with receptors, such as G protein-coupled receptors and ligand-gated ion channels, to induce or inhibit a biological response. When no natural ligands are present, a receptor exists in an equilibrium of inactive (Ri) and active (Ra) conformations. The inactive form does not produce a response, while the active form generates a basal effect known as constitutive activity.
The binding affinity of a drug determines its interaction with...
The binding affinity of a drug determines its interaction with...
3.5K
01:22Activation and Inactivation of G Proteins
12.5K
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
12.5K