Gα16, a G Protein α Subunit Specifically Expressed in Hematopoietic Cells

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein α subunit, Gα16, is specifically expressed in human cells of the hematopoietic lineage. The Gα16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the Gq class of α subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein α subunits, the Gα16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the Gα16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. Gα16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of Gα16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress Gα16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that Gα16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin

Training Culturally Competent Practitioners: Student Reflections On The Process

A major aspect of cultural competence is developing critical self-reflection skills. Critical self-reflection is a culturally competent practice that positions practitioners with the ability to recognize and respond to the influence of power, privilege, and oppression within client interactions. Contributing to the existing literature on cultural competence, this article posits that teaching critical self-reflection is an essential aspect of training culturally competent practitioners. To investigate this connection, researchers qualitatively examined the retrospective accounts of 15 human services students who critically reflected on an assignment: exploring how they were personally impacted by issues of power, privilege, and oppression. Findings unveiled students’ perceptions of developing culturally competent critical self-reflection to be characterized by working through resistance, exploring personal biases, and developing empathy. Implications suggest that instructors seeking to train culturally competent human services practitioners should strategically integrate opportunities for students to work through resistance, explore their existing biases, and develop empathy

Functional Analysis of a Dominant Negative Mutant of Gαi2

The key event in receptor-catalyzed activation of heterotrimeric G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta complexes. We have previously identified a mutation that abolished GTP binding in Galpha(o) (S47C) and demonstrated that the mutant retained the ability to bind beta and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V. Z., Quick, M. W., Aragay, A. M., Davidson, N., Lester, H. A., and Simon, M. I.(1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of Galpha (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the Galpha S48C mutant prevented stimulation of phospholipase C (PLC) beta2 by free beta subunit complexes. This effect of Galpha(i) S48C was not readily reversible in contrast to the inhibitory effect of wild-type Galpha, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta from the G protein heterotrimer. Coexpression of Galpha(i) S48C or the wild-type Galpha also dramatically decreased G-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and Galpha. Activation of PLC via endogenous G(q) or G in the presence of alpha1C adrenergic receptors was similarly attenuated by coexpression of Galpha(i) or Galpha(i) S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type Galpha(i) subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type Galpha(i) inhibitory effect by pertussis toxin can be explained by stabilization of Galpha(i) binding to beta as a result of ADP-ribosylation, while Galpha(i) S48C mutant binds beta irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the Galpha and G-mediated activation of PLC by cotransfected Galpha(i) is the competition between Galpha(i) and Galpha or G for the beta complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta in the integration of signals controlling phosphoinositide release through different Galpha families

The N terminus of phosducin is involved in binding of βγ subunits of G protein

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the βγ subunits of guanine nucleotide-binding proteins (G proteins) (Gβγ) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind Gβγ was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit Gβγ-mediated phospholipase C β_2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-Gβγ interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to Gβγ. In contrast, the C-terminal half failed to bind to Gβγ. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between Gβγ and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residu

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

BACKGROUND: The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. RESULTS: In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. CONCLUSION: Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB

Thermal stability, mechanical properties, and tribological performance of TiAlXN coatings: Understanding the effects of alloying additions

In tribological applications, the degradation of metallic coatings due to oxidation and thermal softening at high temperatures is an issue of increasing concern. Recently, researchers have focused on the development of durable hard coatings that can perform well under elevated temperatures. The alloying of ternary TiAlN coatings with various elements has received considerable attention due to its ability to improve coating properties at high temperatures by solid solution hardening, grain refinement, formation of new phases, diffusion barriers, and self-lubricious tribo-oxides. This paper reviews the microstructure, thermal stability, oxidation behaviour, and mechanical and tribological properties of resultant quaternary TiAlXN coatings (X = Si, Cr, V, Ta and B). The effects of the deposition parameters, chemical composition, high-temperature annealing, and coating architecture on the coating properties are discussed in depth. The properties of quinary TiAlCrSiN coatings are also reviewed to provide a better understanding of the synergistic effects of Si and Cr additions to TiAlN. The maximum hardness and plastic deformation resistance (H/E and H3/E2) of TiAlXN coatings produced by various deposition techniques are compared. This paper provides useful insights into the challenges and future research perspectives of the reviewed coatings

Characterization of G-protein α subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines

Murine Gα14 and Gα15 cDNAs encode distinct α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with Gαq, Gα11, and Gα16, such as the absence of a pertussis toxin ADP-ribosylation site. Gα11 and Gαq are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas Gα15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, Gα11 mRNA is found in all cell lines tested, Gαq is expressed widely but is not found in most T-cell lines, Gα15 is predominantly expressed in myeloid and B-cell lineages, and Gα14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of Gα15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with Gα15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function

Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding

Nucleotide binding properties of the G protein ɑ subunit G_oɑ were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [^(35)S]GTPyS (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G_oɑ cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G_oɑ for GTPyS or Mg^(2+) were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21^(ras) proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPyS binding but retained the capacity to interact with the βy subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G_oɑ increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl^- response, suggesting that this mutation resulted in a dominant negative phenotype

Characterization of a Goα Mutant That Binds Xanthine Nucleotides

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goα mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goα subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoαD273N/Q205L (GoαX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoαX (GoαD273N/Q205L) formed heterotrimers with βγ complexes only in the presence of xanthine diphosphate (XDP), and the binding to βγ was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoαX protein underwent a conformational change similar to that of the activated wild-type Goα. In transfected COS-7 cells, we demonstrate that the interaction between GoαX and βγ occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein α subunit