ABSTRACT
G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the ß(2)-adrenergic receptor (ß(2)AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist-ß(2) adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of ß(2)AR and the C-terminal end of the Gα(s) subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between ß(2)AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, ß(2)AR and GαCT form a stable interaction that is destabilized by carazolol.
Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Molecular Dynamics Simulation , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Adrenergic beta-2 Receptor Agonists/chemistry , Adrenergic beta-2 Receptor Agonists/pharmacology , Binding Sites/drug effects , Crystallography, X-Ray/methods , Diffusion , Isoproterenol/chemistry , Isoproterenol/pharmacology , Ligands , Models, Molecular , Opsins/chemistry , Opsins/metabolism , Propanolamines/chemistry , Propanolamines/pharmacology , Protein Binding/drug effects , Protein Structure, Secondary , Signal Transduction/drug effectsABSTRACT
Heterocyclic dopamine surrogates of types 5 and 7 were synthesized and investigated for their dopaminergic properties. The enantiomerically pure biphenylcarboxamide (S)-5a displayed an outstanding K(i) of 27 pM at the agonist-labeled D(3) receptor and significant selectivity over the D(2) subtype. Measurement of [(35)S]GTPγS incorporation in the presence of a coexpressed PTX-insensitive G(α0-1) subunit indicated highly efficient G-protein coupling. Comparison of ligand efficacy data from cAMP accumulation and [(3)H]thymidine incorporation experiments revealed that ligand biased signaling is exerted by the test compound (S)-5a. Starting from the D(3) crystal structure, a combination of homology modeling and site directed mutagenesis gave valuable insights into the binding mode and the intermolecular origins of stereospecific receptor recognition. According to these data, the superior affinity of the eutomer 5a is caused by the favorable binding energy that results from interaction between the ligand's central ammonium unit and the aspartate residue in position 3.32 of the receptor.
Subject(s)
Pyrazoles/chemistry , Pyrazoles/metabolism , Pyridines/chemistry , Pyridines/metabolism , Receptors, Dopamine D3/metabolism , HEK293 Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Protein Conformation , Pyrazoles/chemical synthesis , Pyridines/chemical synthesis , Receptors, Dopamine D3/chemistry , Receptors, Dopamine D3/genetics , Stereoisomerism , Substrate SpecificityABSTRACT
Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with alpha(1)- and alpha(2)-adrenergic, beta(2)-adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant and had reduced potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. In contrast, Ro10-4548 [RAC-2'-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide], a catechol-like antagonist of the wild-type receptor unexpectedly functions as an agonist of the S5.43A mutant. Other noncatechol ligands had similar properties for mutant and wild-type receptors. This is the first example of a dopamine receptor point mutation selectively changing the receptor's interaction with a specific antagonist to that of an agonist, and together with other data, provides evidence, supported by molecular modeling, that catecholamine-type agonism is induced by different ligand-specific configurations of intermolecular H-bonds with the TM5 conserved serines.