Abstract
G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.
Highlights
We devised a strategy that engineered the trafficking of a GPCR to another cellular compartment and hypothesized that if it took with it its oligomeric partner, this would provide definitive proof of oligomerization and provide a tool to study its dynamics in the cell
By fluorescence resonance energy transfer analysis, we revealed that the D1 and D2 receptors exist in close proximity on the cell surface, presumably within a heterooligomeric complex
We show that incorporating an nuclear localization signal (NLS) into several of the dopamine receptors mediated receptor translocation to the nucleus
Summary
The biophysical techniques utilized to investigate GPCR oligomers, such as bioluminescence resonance energy transfer (14) or fluorescence resonance energy transfer (9, 15), permitted the analysis of receptorreceptor or receptor-protein interactions in situ, within living cells These techniques still have only limited ability to investigate the many aspects of oligomer structure or function that still remain unknown. We and others determined that homodimerization in the rhodopsin-like GPCRs utilizes a transmembrane domain dimer interface (6, 16, 17, 18), and we predicted that this interaction would remain intact during the engineered receptor trafficking This process may permit us to visualize homo- and heterooligomer formation and, using conformationally altered receptor variants, to probe the contribution of the receptor structure to the stability of oligomers. The D1 and D2 receptor heterooligomers displayed novel agonist-induced internalization and trafficking patterns, distinct from that of D1 and D2 receptor homooligomers (26)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
