tailieunhanh - Bioluminescence Recent Advances in Oceanic Measurements and Laboratory Applications Part 7

Tham khảo tài liệu 'bioluminescence recent advances in oceanic measurements and laboratory applications part 7', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 4 Quantitative Assessment of Seven Transmembrane Receptors 7TMRs Oligomerization by Bioluminescence Resonance Energy Transfer BRET Technology Valentina Kubale1 Luka Drinovec2 and Milka Vrecl1 institute of Anatomy Histology Embryology Veterinary Faculty of University in Ljubljana 2Aerosol . Ljubljana Slovenia 1. Introduction Seven transmembrane receptors 7TMRs also designated as G-protein coupled receptors GPCRs form the largest and evolutionarily well conserved family of cell-surface receptors with more than 800 members identified in the human genome. 7TMRs are the targets both for a plethora of endogenous ligands . peptides glycoproteins lipids amino acids nucleotides neurotransmitters odorants ions and photons and therapeutic drugs and transduce extracellular stimuli into intracellular responses mainly via coupling to guanine nucleotide binding proteins G-proteins McGraw Liggett 2006 . These receptors have traditionally been viewed as monomeric entities and only more recent biochemical and biophysical studies have changed this view. The idea that 7TMRs might form dimers or higher order oligomeric complexes has been formulated more than 20 years ago and since then intensively studied. In the last decade bioluminescence resonance energy transfer BRET was one of the most commonly used biophysical methods to study 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on non-radiative transfer of energy between donor and acceptor their intermolecular distance 10 -100 A and relative orientation. Over this period the method has progressed and several versions of BRET have been developed that use different substrates and or energy donor acceptor couples to improve stability and specificity of the BRET signal. This chapter outlines BRET-based approaches to study 7TMRs oligomerization . BRET saturation and competition assays control experiments .