In this study, we examined the mechanisms of intermolecular interaction involved in D2 dopamine receptor dimer formation to develop an understanding of the quaternary structure of G protein-coupled receptors. The potential role of two mechanisms was investigated: disulfide bridges and hydrophobic interactions between transmembrane domains. D2 dopamine receptor oligomers were unaffected by treatment with a reducing agent; however, oligomers of the D1 dopamine receptor dissociated following a similar treatment. This observation suggested that other forces such as hydrophobic interactions were more robust in the D2 receptor than in the D1 receptor in maintaining oligomerization. To elucidate which transmembrane domains were involved in the intermolecular hydrophobic interactions, truncation mutants were generated by successive deletion of transmembrane domains from amino and/or carboxyl portions of the D2 dopamine receptor. Immunoblot analyses revealed that all the fragments were well expressed but only fragments containing transmembrane domain 4 were able to self-associate, suggesting that critical areas for receptor dimerization resided within this transmembrane domain. Disruption of the helical structure of transmembrane domain 4 in a truncated receptor capable of forming dimers interfered with its ability to self-associate; however, a similar disruption of the transmembrane domain 4 helix structure in the full-length receptor did not significantly affect dimerization. These results indicated that there are other sites of interaction involved in D2 receptor oligomer assembly in addition to transmembrane domain 4.

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. Agonistnonbinding 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.G protein-coupled receptors (GPCRs) 3 form dimers and higher order oligomers, as inferred from a large body of evidence garnered from a variety of methodological approaches (1-3), and their static configuration has been visualized by atomic force microscopy in the case of rhodopsin to reveal dimers arranged in clustered rows, with each row accommodating 10 -20 dimers (4, 5). Rhodopsin-related GPCRs function as arrays of oligomers (5, 6) and form complexes with identical or other GPCRs, generating homo-or hetero-oligomers (7-10). The structural details involved in the formation of receptor dimers or oligomers have not been elucidated, with little experimental evidence for fundamental questions, such as their behavior at the cell surface, whether the oligomers remain intact or separate, and if homooligomers and heterooligomers behave differently. That the oligomerized GPCR structures modulate the properties and conformations of the individual constituent receptors involved has been shown for -and ␦-opioid receptor heterooligomers (8,11,12) and D1 and D2 dopamine receptor heterooligomers (13). In these structures, the ligand binding properties and/or the coupling ...

Available symptomatic therapies for the treatment of Alzheimer's disease (AD) have been based on known neurotransmitter dysfunctions associated with the illness. The second-generation cholinesterase inhibitors and the N-methyl D-aspartate receptor antagonist memantine have been widely prescribed and studied. Meta-analyses of these therapies were reviewed, focusing on effectiveness and tolerability. Although many of the meta-analyses demonstrate statistically significant improvements, some question if these benefits are sufficient to justify their current widespread and protracted use. This has spurred the development of new disease-modifying therapies that aim to have a greater impact on this debilitating illness.