Ligand-Dependent Conformations and Dynamics of the Serotonin 5-HT2A Receptor Determine Its Activation and Membrane-Driven Oligomerization Properties

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization

Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

<p>Abstract</p> <p>Background</p> <p>A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β₂-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling.</p> <p>Results</p> <p>C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface.</p> <p>Conclusions</p> <p>We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer.</p

Crystal structure of a photoactivated deprotonated intermediate of rhodopsin

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors

Hallucinogen actions on 5-HT receptors reveal distinct mechanisms of activation and signaling by G protein-coupled receptors

We review the effect of some key advances in the characterization of molecular mechanisms of signaling by G protein-coupled receptors (GPCRs) on our current understanding of mechanisms of drugs of abuse. These advances are illustrated by results from our ongoing work on the actions of hallucinogens on serotonin (5-HT) receptors. We show how a combined computational and experimental approach can reveal specific modes of receptor activation underlying the difference in properties of hallucinogens compared with nonhallucinogenic congeners. These modes of activation—that can produce distinct ligand-dependent receptor states—are identified in terms of structural motifs (SM) in molecular models of the receptors, which were shown to constitute conserved functional microdomains (FM). The role of several SM/FMs in the activation mechanism of the GPCRs is presented in detail to illustrate how this mechanism can lead to ligand-dependent modes of signaling by the receptors. Novel bioinformatics tools are described that were designed to support the quantitative mathematical modeling of ligand-specific signaling pathways activated by the 5-HT receptors targeted by hallucinogens. The approaches for mathematical modeling of signaling pathways activated by 5-HT receptors are described briefly in the context of ongoing work on detailed biochemical models of 5-HT2A, and combined 5-HT2A/5-HT1A, receptor-mediated activation of the MAPK 1,2 pathway. The continuing need for increasingly more realistic representation of signaling in dynamic compartments within the cell, endowed with spatio-temporal characteristics obtained from experiment, is emphasized. Such developments are essential for attaining a quantitative understanding of how the multiple functions of a cell are coordinated and regulated, and to evaluate the specifics of the perturbations caused by the drugs of abuse that target GPCRs