Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1–8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.

Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.

The three members of the pocket proteins family share the ability to repress E2F activity through recruitment of a histone deacetylase

The transcription factor E2F plays a major role in cell cycle control in mammalian cells. E2F binding sites, which are present in the promoters of a variety of genes required for S phase, shift from a negative to a positive role in transcription at the commitment point, a crucial point in G1 that precedes the G1/S transition. Before the commitment point, E2F activity is repressed by members of the pocket proteins family. This repression is believed to be crucial for the proper control of cell growth. We have previously shown that Rb, the founding member of the pocket proteins family, represses E2F1 activity by recruiting the histone deacetylase HDAC1. Here, we show that the two other members of the pocket proteins family, p107 and p130, also are able to interact physically with HDAC1 in live cells. HDAC1 interacts with p107 and Rb through an “LXCXE”-like motif, similar to that used by viral transforming proteins to bind and inactivate pocket proteins. Indeed, we find that the viral transforming protein E1A competes with HDAC1 for p107 interaction. We also demonstrate that p107 is able to interact simultaneously with HDAC1 and E2F4, suggesting a model in which p107 recruits HDAC1 to repress E2F sites. Indeed, we demonstrate that histone deacetylase activity is involved in the p107- or p130-induced repression of E2F4. Taken together, our data suggest that all members of the E2F family are regulated in early G1 by similar complexes, containing a pocket protein and the histone deacetylase HDAC1.

Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix–turn–helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1

We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.

Three Drought-Responsive Members of the Nonspecific Lipid-Transfer Protein Gene Family in Lycopersicon pennellii Show Different Developmental Patterns of Expression1

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

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.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

A heterodimeric DNA polymerase: Evidence that members of Euryarchaeota possess a distinct DNA polymerase

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.