Konstantin Levay

Complexes of the G Protein Subunit Gβ5 with the Regulators of G Protein Signaling RGS7 and RGS9 CHARACTERIZATION IN NATIVE TISSUES AND IN TRANSFECTED CELLS

A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gβ5-RGS and their coupled expression argue that in vivo, Gβ5and Gγ-like domain-containing RGSs only exist as heterodimers. Native Gβ5-RGS7 did not co-precipitate or co-purify with Gαo or Gαq; nor did Gβ5 L-RGS9 with Gαt. However, in transfected cells, RGS7 and Gβ5-RGS7 inhibited Gαq-mediated Ca2+ response to muscarinic M3 receptor activation. Thus, Gβ5-RGS dimers differ from other RGS proteins in that they do not bind to Gα with high affinity, but they can still inhibit G protein signaling.

Structural Basis for Calcium-induced Inhibition of Rhodopsin Kinase by Recoverin

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca2+-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca2+-bound recoverin in the absence of target (<1.8Å root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca2+ is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca2+-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca2+-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca2+.

Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

Evidence for Multiple Phototransduction Pathways in a Reef-Building Coral

Photosensitive behaviors and circadian rhythms are well documented in reef-building corals and their larvae, but the mechanisms responsible for photoreception have not been described in these organisms. Here we report the cloning, immunolocalization, and partial biochemical characterization of three opsins and four G proteins expressed in planulae of the Caribbean elkhorn coral, Acropora palmata. All three opsins (acropsins 1–3) possess conserved seven-pass transmembrane structure, and localize to distinct regions of coral planulae. Acropsin 1 was localized in the larval endoderm, while acropsin 2 was localized in solitary cells of the ectoderm. These rod-like cells displayed a remarkably polarized distribution, concentrated in the aboral end. We also cloned four A. palmata G protein alpha subunits. Three were homologs of vertebrate Gi, Go, and Gq. The fourth is presumably a novel G protein, which displays only 40% identity with the nearest known G protein, and we termed it Gc for “cnidarian”. We show that Gc and Gq can be activated by acropsins in a light-dependent manner in vitro. This indicates that at least acropsins 1 and 3 can form functional photoreceptors and potentially may play a role in color preference during settlement, vertical positioning and other light-guided behaviors observed in coral larvae.

Targeted deletion of one or two copies of the G protein β subunit Gβ5 gene has distinct effects on body weight and behavior in mice

We investigated the physiological role of Gβ5, a unique G protein β subunit that dimerizes with regulators of G protein signaling (RGS) proteins of the R7 family instead of Gβ. Gβ5 is essential for stability of these complexes, so that its knockout (KO)causes degradation of the entire Gβ5‐R7 family. We report that the Gβ5‐KO mice remain leaner than the wild type (WT) throughout their lifetime and are resistant to a high‐fat diet. They have a 5‐fold increase in locomotor activity, increased thermogenesis, and lower serum insulin, all of which correlate with a higher level of secreted epinephrine. Heterozygous (HET) mice are 2‐fold more active than WT mice. Surprisingly, with respect to body weight, the HET mice display a phenotype opposite to that of the KO mice: by the age of 6 mo, they are ≥15% heavier than the WT and have increased adiposity, insulin resistance, and liver steatosis. These changes occur in HET mice fed a normal diet and without apparent hyperphagia, mimicking basic characteristics of human metabolic syndrome. We conclude that even a partial reduction in Gβ5‐R7 level can perturb normal animal metabolism and behavior. Our data on Gβ5 haploinsufficient mice may explain earlier observations of genetic linkage between R7 family mutations and obesity in humans.—Wang, Q., Levay, K., Chanturiya, T., Dvoriantchikova, G., Anderson, K. L., Bianco, S. D. C., Ueta, C. B., Molano, R. D., Pileggi, A., Gurevich, E. V., Gavrilova, O., and Slepak, V. Z. Targeted deletion of one or two copies of the G protein β subunit Gβ5 gene has distinct effects on body weight and behavior in mice. FASEB J. 25, 3949–3957 (2011). www.fasebj.org

Expression of Olfactory Signaling Genes in the Eye

Purpose To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. In situ hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment.

Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats.

RGS proteins regulate G protein‐mediated signalling pathways through direct interaction with the Gα subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein β subunit Gβ5 via a characteristic Gγ‐like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7‐Gβ5 complex in inflammatory cells, we performed immunocytochemistry, RT‐PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non‐stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF‐α In addition, RGS7‐positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7‐Gβ5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post‐traumatic inflammatory responses in the central nervous system.

Subcellular localization of Regulator of G protein Signaling RGS7 complex in neurons and transfected cells

J. Neurochem. (2012) 122, 568–581.

Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage

Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

Regulation of Cop9 signalosome activity by the EF-hand Ca2+-binding protein tescalcin

ABSTRACT The Ca2+-binding protein tescalcin is known to be involved in hematopoietic cell differentiation; however, this mechanism is poorly understood. Here, we identify CSN4 (subunit 4 of the COP9 signalosome) as a novel binding partner of tescalcin. The COP9 signalosome (CSN) is a multiprotein complex that is essential for development in all eukaryotes. This interaction is selective, Ca2+-dependent and involves the PCI domain of CSN4 subunit. We then investigated tescalcin and CSN activity in human erythroleukemia HEL and promyelocytic leukemia K562 cells and find that phorbol 12-myristate 13-acetate (PMA)-induced differentiation, resulting in the upregulation of tescalcin, coincides with reduced deneddylation of cullin-1 (Cul1) and stabilization of p27Kip1 – molecular events that are associated with CSN activity. The knockdown of tescalcin led to an increase in Cul1 deneddylation, expression of F-box protein Skp2 and the transcription factor c-Jun, whereas the levels of cell cycle regulators p27Kip1 and p53 decreased. These effects are consistent with the hypothesis that tescalcin might play a role as a negative regulator of CSN activity towards Cul1 in the process of induced cell differentiation.