Structural dynamics of G protein-coupled receptor monomers and oligomers: Insights from the beta2-adrenergic receptor

Using a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), we studied various aspects of receptor monomeric structure and dynamics, as well as studies to understand the mechanisms of receptor oligomerization.;In our initial experiments using purified, monomeric beta2AR, we generated several mutants to address structural aspects of the C-terminus using Fluorescence Resonance Energy Transfer (FRET). The C-terminus plays a role in ligand-dependent protein-protein interactions such as arrestin desensitization/signaling and phosphorylation by GRK. Thus, we introduced tetracysteine motifs (CCPGCC) at the proximal C-terminus (from residues 351-356) or distal C-terminus (starting at the last residue 413) that are capable of specifically binding to Fluoresceine Arsenical Helix binders (FlAsH), the donor fluorophore for our intramolecular FRET studies.;Immunization of mice with beta2AR in liposomes resulted in the production of nine monoclonal antibodies, five which bound the intracellular domain of the receptor and four that bound the extracellular domain. Only two of the nine monoclonal antibodies (Ab 5 and Ab 9) were able to recognize a 3-dimensional epitope as assessed by immunoblotting to denatured beta 2AR. Because Ab 5 also recognized an intracellular 3-dimensional epitope, it was chosen to generate Fabs for crystal trials of the beta2AR. Vapor phase diffusion, using ammonium sulfate as the precipitant, resulted in diffraction quality crystals of Fab 5 complexed to the beta2AR (beta2AR-Fab5). Optimization of the crystallizing conditions resulted in a subsequent high-resolution structure (3.4 A) of monomeric beta 2AR bound to an inverse agonist, the first for a GPCR activated by a diffusible ligand.;We developed methods for studying the quaternary structure of purified beta 2AR to understand oligomeric structural aspects of this prototypical GPCR. Using a minimal cysteine beta2AR construct (Delta5-beta 2AR), three single-reactive cysteine mutants were generated for FRET studies: T66C (Delta5-beta2AR-T66C) in intracellular loop 1 (ICL1); A265C (Delta5-beta2AR-A265C) at the base of TM6; and R333C (Delta5-beta2AR-R333C) in helix 8 (H8). Using these three beta 2AR mutants, we could obtain maximal spatial distribution from our donor (Cy3-maleimide) and acceptor (Cy5-maleimide) fluorophores as well as infer into the orientation of protomers in an oligomeric assembly using FRET. Reconstitution of these purified receptors into a model DOPC/cholesterol hemisuccinate lipid bilayer yielded an even distribution of fully functional receptors in vesicles, mostly in an outside-out orientation as determined by four independent biochemical techniques. Intermolecular FRET analysis revealed that the beta2AR forms specifically oriented oligomers upon reconstitution into a lipid bilayer, independent of other cellular proteins and that TM6-TM6 interactions are the furthest from the regions investigated. Overcrowding the lipid vesicle membrane with receptors was not the cause of FRET since reconstitution with a 10-fold higher lipid-to-receptor ratio resulted in insignificant changes in FRET. Furthermore, saturation FRET experiments were used in order to address the specificity of interactions. It follows that if two proteins have an affinity for one another, a saturable amount of FRET should be observed, whereas randomly colliding proteins will follow a quasi-linear regression. Indeed, all three single-reactive cysteine beta2AR mutants demonstrated saturable amounts of FRET. We then used a mathematical model described previously to generate theoretical FRET saturation curves for dimers, trimers, tetramers and higher-order oligomers. Our experimental results demonstrated that the beta 2AR more closely follows the theoretical curve for a tetramer, suggesting that beta2ARs can form specific tetramers in the absence of other cellular proteins in lipid bilayers. This allowed us to propose a model of a beta2AR tetramer where TM6s are on the periphery of the tetramer, due to the consistently lower FRET in the TM6-TM6 pair.;Taken together, the investigations presented offer an in-depth view of the structural dynamics of a prototypical GPCR, the beta2AR, and provide novel avenues to explore receptor activation and inactivation at the molecular and supramolecular level. (Abstract shortened by UMI.)...