Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells). Single-lymphocyte recording techniques failed to identify an ionic current in response to applied acetylcholine in either T-cells or B-cells. These results clearly demonstrate the presence of these nicotinic receptor subunits within several populations of human lymphocytes, implicating their role in the immune response. However, a lack of demonstrated response to applied acetylcholine using standard single-cell recording techniques suggests a physiology different than that seen in hair cells of the inner ear.     

Haplotypes of alpha-globin-gene in the Saudi population--the triplicated gene (alpha alpha alpha anti 3.7/)

Using restriction endonucleases Bam HI, Bg1 II, Hpa I, Hind III and Xba I, the alpha alpha alpha anti 3.7/ haplotype was studied in the Saudi population. This paper presents the frequency of the alpha gene haplotype alpha alpha alpha anti 3.7/ in different regions of Saudi Arabia, where thalassaemia occurs at a high frequency.     

Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.     

Isolation and characterization of CNBr peptides of human (alpha 1 (III) )3 collagen and tissue distribution of (alpha 1 (I) )2 alpha 2 and (alpha 1 (III) )3 collagens.

[Alpha 1(III)]3 collagen was solubilized by pepsin digestion of normal human placental membranes and was purified by differential salt precipitation and carboxymethylcellulose chromatography. This collagen was digested with CNBr, and the resultant nine peptides were isolated and characterized. The chains are cross-linked by cysteinyl residues in the COOH-terminal peptide. Isolation of peptides derived from CNBr digestion of insoluble tissues was used as an assay for the presence of [alpha 1(I)]2alpha 2 and [alpha 1(III)]3 collagens. Both types are present in human skin, intestine, liver, spleen, kidney, lung, aorta, umbilical cord, placental membranes, and myocardium. Bone and tendon contain [alpha 1(I)]2alpha 2 collagen but, unlike the other tissues, lack [alpha 1(III)]3 collagen. Both [alpha 1(I)]2alpha 2 and[alpha 1(III)]3 collagens are present in scars of human skin, myocardium, tendon, and liver and of rabbit skin. The degree of hydroxylation of proline was 4 to 5% lower in the same peptides in skin, bone, and tendon than in the other tissues. The degree of hydroxylation of lysine in the same peptides derived from different tissues varied more widely.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity

Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)(2)α4 nAChRs.     

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.     

Peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) enhances the thyroid hormone induction of carnitine palmitoyltransferase I (CPT-I alpha)

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-controlling step in the pathway of mitochondrial fatty acid oxidation. Thyroid hormone will stimulate the expression of the liver isoform of CPT-I (CPT-I alpha). This induction of CPT-I alpha gene expression requires the thyroid hormone response element in the promoter and sequences within the first intron. The peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) is a coactivator that promotes mitochondrial biogenesis, mitochondrial fatty acid oxidation, and hepatic gluconeogenesis. In addition, PGC-1 alpha will stimulate the expression of CPT-I alpha in primary rat hepatocytes. Here we report that thyroid hormone will increase PGC-1 alpha mRNA and protein levels in rat hepatocytes. In addition, overexpression of PGC-1 alpha will enhance the thyroid hormone induction of CPT-I alpha indicating that PGC-1 alpha is a coactivator for thyroid hormone. By using chromatin immunoprecipitation assays, we show that PGC-1 alpha is associated with both the thyroid hormone response element in the CPT-I alpha gene promoter and the first intron of the CPT-I alpha gene. Our data demonstrate that PGC-1 alpha participates in the stimulation of CPT-I alpha gene expression by thyroid hormone and suggest that PGC-1 alpha is a coactivator for thyroid hormone.     

Triple alpha-genes (alpha alpha alpha anti3.7) in a patient with sickle cell anaemia.

This paper reports the case of a 17-year-old male student from the Jaizan area in south-western Saudi Arabia who had sickle cell anaemia and possessed three alpha-genes on one chromosome (alpha alpha alpha anti3.7) and two on the other. The clinical manifestations were severe, with frequent blood transfusion requirements and frequent episodes of painful crises, severe anaemia and tissue involvement. In comparison with age and sex-matched sickle cell anaemia patients with one alpha-gene deletion (-alpha/alpha alpha), or a normal alpha-gene arrangement (alpha alpha/alpha alpha), a more severe disease presentation was obvious in the propositus. It is suggested that with the surplus alpha-globin chains, more severe haematological and clinical abnormalities occur, these influence the phenotypic expression of sickle cell anaemia. However, more patients with this type of gene arrangement must be studied before a definite conclusion can be reached regarding the influence of excess alpha-globin chains on the presentation of sickle cell anaemia.     

Growth arrest and DNA damage-45 alpha (GADD45alpha)

Regulation of cell cycle and growth is integral for cell survival. The intricate mechanisms that control proliferation and cell cycle are numerous. The growth arrest and DNA damage (GADD)-inducible gene family is often up-regulated in response to various environmental stresses and drug therapies. GADD45alpha was the first stress-inducible gene determined to be up-regulated by p53 and is also a target for the p53 homologues, p63 and p73. When GADD45alpha is deleted or repressed, cells show uncontrolled proliferation. Furthermore, decreased GADD45alpha expression is also considered a survival mechanism, as cancer cells without this control can evade the apoptotic pathway leading to increased tumourigenesis. Drug therapies can act to directly or indirectly up-regulate GADD45alpha and promote apoptosis. As GADD45alpha is an essential component of many metabolic pathways that control proliferating cancer cells, it presents itself as an emerging drug target worthy of further investigation.     

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.