Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gß and Gγ Subunits in C57Bl6/J Brain Synaptosomes.

Gßγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gß and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gß and Gγ subunits selectively interact to form unique Gßγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gßγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gß and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gß and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gß and Gγ subunits and their subcellular localizations. For example, Gß1 was mostly localized at the membrane while Gß2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gß and Gγ subunits may affect the Gßγ dimerization and Gßγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gß and Gγ subunits but also new insights into the in vivo distribution of Gß and Gγ subunits, and Gßγ dimer assembly in normal brain function.

Gßγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.

Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gßγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gßγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gßγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gßγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gßγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gßγ for SNARE-binding sites in lipid environments. Mutant Gßγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gßγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gßγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gß and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gßγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gßγ-SNARE interaction and show that the target of Gßγ, downstream of VGCC, is the membrane-embedded SNARE complex.

Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine.

In heart failure, the ß-adrenergic receptors (ßARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, 14as, with an IC50 of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, 14as showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC50 for over 7 h. Three of these inhibitors, including 14as, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of these inhibitors.

Structure-Based Design, Synthesis, and Biological Evaluation of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors.

G protein-coupled receptors (GPCRs) are central to many physiological processes. Regulation of this superfamily of receptors is controlled by GPCR kinases (GRKs), some of which have been implicated in heart failure. GSK180736A, developed as a Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor, was identified as an inhibitor of GRK2 and co-crystallized in the active site. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Takeda103A, a library of hybrid inhibitors was developed. This campaign produced several compounds possessing high potency and selectivity for GRK2 over other GRK subfamilies, PKA, and ROCK1. The most selective compound, 12n (CCG-224406), had an IC50 for GRK2 of 130 nM, >700-fold selectivity over other GRK subfamilies, and no detectable inhibition of ROCK1. Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors.

Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2-Gß1γ2 Complex.

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gßγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4',5'-bisphosphate (PIP2). We hypothesized that PIP2 may favor a distinct orientation of the GRK2-Gßγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2-Gßγ complex and Gßγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP2 affects the membrane orientation of the GRK2-Gß1γ2 complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gß1γ2 exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP2-induced orientation of the GRK2-Gß1γ2 complex is therefore most likely caused by specific interactions between PIP2 and the GRK2 PH domain. Thus, PIP2 not only helps recruit GRK2 to the membrane but also "fine tunes" the orientation of the GRK2-Gßγ complex so that it is better positioned to phosphorylate activated GPCRs.

Bifunctional ligands allow deliberate extrinsic reprogramming of the glucocorticoid receptor.

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics.

The in vivo role of androgen receptor SUMOylation as revealed by androgen insensitivity syndrome and prostate cancer mutations targeting the proline/glycine residues of synergy control motifs.

The androgen receptor (AR) mediates the effects of male sexual hormones on development and physiology. Alterations in AR function are central to reproductive disorders, prostate cancer, and Kennedy disease. AR activity is influenced by post-translational modifications, but their role in AR-based diseases is poorly understood. Conjugation by small ubiquitin-like modifier (SUMO) proteins at two synergy control (SC) motifs in AR exerts a promoter context-dependent inhibitory role. SC motifs are composed of a four-amino acid core that is often preceded and/or followed by nearby proline or glycine residues. The function of these flanking residues, however, has not been examined directly. Remarkably, several AR mutations associated with oligospermia and androgen insensitivity syndrome map to Pro-390, the conserved proline downstream of the first SC motif in AR. Similarly, mutations at Gly-524, downstream of the second SC motif, were recovered in recurrent prostate cancer samples. We now provide evidence that these clinically isolated substitutions lead to a partial loss of SC motif function and AR SUMOylation that affects multiple endogenous genes. Consistent with a structural role as terminators of secondary structure elements, substitution of Pro-390 by Gly fully supports both SC motif function and SUMOylation. As predicted from the functional properties of SC motifs, the clinically isolated mutations preferentially enhance transcription driven by genomic regions harboring multiple AR binding sites. The data support the view that alterations in AR SUMOylation play significant roles in AR-based diseases and offer novel SUMO-based therapeutic opportunities.