Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.

Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.

A functional Rev-erb alpha responsive element located in the human Rev-erb alpha promoter mediates a repressing activity.

Rev-erb alpha belongs to the nuclear receptor superfamily, which contains receptors for steroids, thyroid hormones, retinoic acid, and vitamin D, as well as "orphan" receptors. No ligand has been found for Rev-erb alpha to date, making it one of these orphan receptors. Similar to some other orphan receptors, Rev-erb alpha has been shown to bind DNA as a monomer on a specific sequence called a Rev-erb alpah responsive element (RevRE), but its transcriptional activity remains unclear. In this paper, we characterize a functional RevRE located in the human Rev-erb alpha promoter itself. We also present evidence that (i) Rev-erb alpha mediates transcriptional repression of its own promoter in vitro, (ii) this repressing effect strictly depends on the binding of Rev-erb alpha to its responsive element and is transferable to a heterologous promoter; and (iii) Rev-erb alpha binds to this responsive sequence as a homodimer.

Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha.

A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV. Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure. Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml. When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml. Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity. Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G -->V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml. These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation.

Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.

Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

Golgi alpha-mannosidase II (alpha-MII) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22.