Final Report Summary - ADORA (Structural and Functional Studies of Human Adenosine A2A Receptor G protein Complex: Toward Understanding GPCR Activation)
A major objective of our project is to resolve the crystal structure of the non-G protein pathway mediator in complex with full-length adenosine A2A receptor and/or receptor domains. Interestingly, it has been reported that the carboxyl terminal part of the adenosine A2A receptor is responsible for G protein-independent signal transduction. While interactions of A2A with several signalling partners are known at a functional and cellular level in vivo and in vitro, little is known at protein and atomic levels. Our long-term goal is to understand the protein–protein interactions and the conformational changes that accompany them at an atomic level, either by using X-ray crystallography (structure) or NMR (interactions, populations, time and distances). Questions relating to the strength, dynamics, stoichiometry and post-translational modifications required for these interactions will also be addressed. In addition, one goal is to obtain better knowledge of long-distance effects of the interactions between the receptor’s extracellular ligand-binding pocket and the intracellular recognition part.
We work simultaneously with wild-type adenosine A2A receptor, truncated receptor (different delta-C) and its large carboxyl terminal domain, of which structural data is currently missing. The main progress has been achieved with A2A-ct subproject, while lately we have been focusing working with the wild-type (wt) receptor. We have now development purification and interaction assays for following interaction partners at a protein level: CaM, alpha-actinin 1, Arf nucleotide site opener (ARNO) and Arf6. We are currently carrying out further work on expression and purification methods for NECAB2 and dopamine D2 3rd intracellular loop domain. For A2A-ct, we started the project by cloning the recombinant DNA constructs required for protein production and optimized the protein expression and purification for each construct separately. We continued by biophysical characterization of A2A-ct, and experimentally verified it’s partially unfolded nature. Far-UV circular dichroism (Far-UV CD) spectrum of the A2A-ct resembles the typical spectrum of disordered protein, and the single tryptophan in the middle of the A2A-ct is in the water environment supporting the far-UV CD results. We saw the A2A-ct/CaM binding on native polyacrylamide gel electrophoresis (native-PAGE) and analytical size exclusion chromatography. The binding requires calcium and A2A-ct amino acids 293-320, since deletion of these amino acids abolished CaM binding. We further characterized the interaction by isothermal titration calorimetry, and obtained a titration curve corresponding 1:1 binding with dissociation constant (Kd) of 100 nM. Finally, we determined the solution structures of A2A-ct alone and in a complex with CaM by small angle X-ray scattering (SAXS). A2A-ct has an extended tubular-like conformation in solution.
CaM binds to one end of the A2A-ct, as an extra bulkier extension appears in the model in the presence of Ca2+/CaM. Most probably CaM binds to the arginine-rich N-terminal part of the A2A-ct next to the cell membrane. We have isotope labeled A2A-ct for NMR. Nearly complete 1H, 13C and 15N NMR assignments have been obtained for A2A-ct. The CaM: A2A-ct interaction will be further studied by NMR.