Third-party bioluminescence resonance energy transfer indicates constitutive association of membrane proteins: application to class a g-protein-coupled receptors and g-proteins.
Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that beta2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.
Kuravi
Sudhakiranmayi
S
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia, USA.
Lan
Tien-Hung
TH
Barik
Arnab
A
Lambert
Nevin A
NA
D004735:Energy Transfer / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D008163:Luminescent Measurements / Q000379:methods*; D008565:Membrane Proteins / Q000502:physiology*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go