A small molecule enhances arrestin-3 binding to the β-adrenergic receptor.

G protein-coupled receptor (GPCR) signaling is terminated by arrestin binding to a phosphorylated receptor. Binding propensity has been shown to be modulated by stabilizing the pre-activated state of arrestin through point mutations or C-tail truncation. Here, we hypothesize that pre-activated rotated states can be stabilized by small molecules, and this can promote binding to phosphorylation-deficient receptors, which underly a variety of human disorders.

Downstream signalling of the disease-associated mutations on GPR56/ADGRG1.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor (GPCR) and mutations on this receptor cause cortical malformation due to the over-migration of neural progenitor cells on brain surface. At pial surface, GPR56 interacts with collagen III, induces Rho-dependent activation through Gα and inhibits the neuronal migration. In human glioma cells, GPR56 inhibits cell migration through Gα -dependent Rho pathway.

Fast Screening of Protein-Protein Interactions Using Förster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells.

Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells.

SMN loss dysregulates microtubule-associated proteins in spinal muscular atrophy model.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease caused by the absence of survival motor neuron (SMN) protein. SMN loss results in impairments of the cytoskeleton, including microtubules and regulatory proteins. However, the contribution of microtubule-associated proteins (MAPs) to microtubule dysregulations in SMA is not fully understood.

Ste2p Under the Microscope: the Investigation of Oligomeric States of a Yeast G Protein-Coupled Receptor.

Oligomerization of G protein-coupled receptors (GPCRs) may play important roles in maturation, internalization, signaling, and pharmacology of these receptors. However, the nature and extent of their oligomerization is still under debate. In our study, Ste2p, a yeast mating pheromone GPCR, was tagged with enhanced green fluorescent protein (EGFP), mCherry, and with split florescent protein fragments at the receptor C-terminus.

Allergen fragrance molecules: a potential relief for COVID-19.

Background: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today.

Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit.

The γ-Aminobutyric acid type A receptors (GABAARs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABAAR subunits tagged with the green fluorescent protein (GFP), a 26.9 kDa protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.

Three dimensional structure prediction of panomycocin, a novel Exo-β-1,3-glucanase isolated from Wickerhamomyces anomalus NCYC 434 and the computational site-directed mutagenesis studies to enhance its thermal stability for therapeutic applications.

Panomycocin is a naturally produced potent antimycotic/antifungal protein secreted by the yeast Wickerhamomyces anomalus NCYC 434 with an exo-β-1,3-glucanase activity. In this study the three dimensional structure of panomycocin was predicted and the computational site-directed mutagenesis was performed to enhance its thermal stability in liquid formulations over the body temperature for topical therapeutic applications. Homology modeling was performed with MODELLER and I-TASSER.

GPCR-Gα protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Gα protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain.

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor).

The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane.

Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus.

GPCRsort-responding to the next generation sequencing data challenge: prediction of G protein-coupled receptor classes using only structural region lengths.

Next generation sequencing (NGS) and the attendant data deluge are increasingly impacting molecular life sciences research. Chief among the challenges and opportunities is to enhance our ability to classify molecular target data into meaningful and cohesive systematic nomenclature. In this vein, the G protein-coupled receptors (GPCRs) are the largest and most divergent receptor family that plays a crucial role in a host of pathophysiological pathways.

Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning.

The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person's history of tobacco use and his or her susceptibility to Parkinson's disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker.

Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery.

The acronym SePhaChARNS, for "selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry," is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes "upregulation" of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning.

Nicotine normalizes intracellular subunit stoichiometry of nicotinic receptors carrying mutations linked to autosomal dominant nocturnal frontal lobe epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (alpha4)(3)(beta2)(2) versus (alpha4)(2)(beta2)(3) subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in alpha4, V287L, and V287M in beta2. alpha4 and beta2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops.

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined.

Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.

The alpha-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228-Leu248) to intensive random mutagenesis and screened multiply substituted alleles for receptor function.

Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors.