Co-transfected COS-7 Cells Research Articles

Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya.

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.

CD40 Ligand Mutants Responsible for X-linked Hyper-IgM Syndrome Associate with Wild Type CD40 Ligand

CD40 ligand (CD40L) is a 33-kDa type II membrane glycoprotein mainly expressed on activated CD4(+) T cells in trimeric form. When it is mutated, the clinical consequences are X-linked hyper-IgM syndrome (XHIM), a primary immunodeficiency disorder characterized by low levels of IgG, IgA, and elevated or normal levels of IgM. Mutated CD40L can no longer bind CD40 nor provide signals for B cells to proliferate and to switch from IgM to other immunoglobulin isotypes. When considering gene therapy for XHIM, it is important to address the possibility that the mutated CD40L associates with transduced wild type CD40L, and as a consequence, immune reconstitution is not attained. In this study, we demonstrate that the various mutated CD40L species we have identified in patients with XHIM, including both full-length and truncated mutants, associate with wild type CD40L on the cell surface of co-transfected COS cells. The association between wild type and mutated CD40L was also observed in CD4(+) T cell lines established from XHIM patients with leaky splice site mutations. The clinical phenotype of these patients suggests that this association between wild type and mutated CD40L species may result in less efficient cross-linking of CD40.

Novel, Missense, and Loss-of-Function Mutations in the Sodium/Iodide Symporter Gene Causing Iodide Transport Defect in Three Japanese Patients

Iodide transport defect is a disorder affecting the active transport of iodide, an essential step in the synthesis of thyroid hormones. We have identified novel germ-line mutations in the Na+/I− symporter (NIS) gene from these Japanese patients with iodide transport defect. One patient had a compound heterozygous mutation of T354P/G93R (Gly93 → ARg [GGC → CGC]), and two sibling patients had a homozygous mutation of G543E (Gly543 → Glu [CGA → GAA]). G93R and G543E, two novel mutations, are located in the 3rd and 12th transmembrane domains of NIS which are encoded by exons 1 and 13, respectively. The NIS mutants carring these mutations had minimal iodide uptake activity when expressed in COS-7 cells, confirming that the identified mutations are the direct cause of the iodide transport defect in these patients. Genotyping of unaffected family members and functional assays of co-transfected COS-7 cells indicate that expression of one normal NIS allele in the heterozygote (T354P, G93R, or G543E) is sufficient to maintain active iodide uptake activity. Thus, one of these NIS mutants acts as a dominant-negative mutant.

Modification of Phosphatidylinositol 3-Kinase SH2 Domain Binding Properties by Abl- or Lck-mediated Tyrosine Phosphorylation at Tyr-688

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.

Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

Molecular basis of receptor/G protein coupling selectivity studied by coexpression of wild type and mutant m2 muscarinic receptors with mutant G alpha(q) subunits.

The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.

Regulation of the Low Molecular Weight Phosphotyrosine Phosphatase by Phosphorylation at Tyrosines 131 and 132

Activation of resting T lymphocytes is initiated by rapid but transient tyrosine phosphorylation of a number of cellular proteins. Several protein tyrosine kinases and protein tyrosine phosphatases are known to be important for this response. Here we report that normal T lymphocytes express the B isoform of low molecular weight protein tyrosine phosphatase B (LMPTP-B). The cDNA was cloned from Jurkat T cells, and an antiserum was raised against it. LMPTP immunoprecipitated from resting Jurkat T cells was found to be tyrosine phosphorylated. On stimulation of the cells through their T cell antigen receptor, the phosphotyrosine content of LMPTP-B declined rapidly. In co-transfected COS cells, Lck and Fyn caused phosphorylation of LMPTP, whereas Csk, Zap, and Jak2 did not. Most of the phosphate was located at Tyr-131, and some was also located at Tyr-132. Incubation of wild-type LMPTP with Lck and adenosine 5'-O-(thiotriphosphate) caused a 2-fold increase in the activity of LMPTP. Site-directed mutagenesis showed that Tyr-131 is important for the catalytic activity of LMPTP, and that thiophosphorylation of Tyr-131, and to a lesser degree Tyr-132, is responsible for the activation.

Role of Gene Overlap in the Regulation of mRNA Translation for Mitochondrial Cytochrome P-450c27/25 in the Rat

Previously published results have revealed sequence complementarity between the 5'-terminal regions of mRNAs for hepatic mitochondrial cytochrome P-450c27/ 25 (c27/25) and serine protease inhibitors (SPI) and predicted a role for this sequence overlap in both the regulation of c27/25 mRNA transcription and translation. The possibility that c27/25 mRNA forms an RNA duplex with complementary sequences of SPI mRNAs in vivo was demonstrated in the rat liver and COS-1 cells cotransfected with c27/25 and SPI2.1 plasmids. Quantitative evaluation of RNA duplex in COS-1 cells revealed that most of the c27/25 mRNA exists in duplex form when SPI2.1 mRNA was present at 5-10-fold that of c27/25 mRNA, a ratio comparable to that observed between these two RNAs in the liver. In cotransfected COS-1 cells with the same ratio of mRNAs, highly significant inhibition of the c27/25 mRNA translation (66-75%) was observed, while its transcription remained unaffected. The partial inhibition of c27/25 mRNA translation, even when most of it exists in duplex form, suggests that RNA duplex is undergoing some type of cytoplasmic processing to disengage c27/25 mRNA and make it available for translation. These results imply that abundant endogenous SPI RNAs are able to regulate the c27/25 gene expression.

Identification of a binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain of RNA polymerase II.

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD) in vitro. Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. We show here that a CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes the Km 0.4 microM to increase by 2 orders of magnitude. Direct binding of the CTD-ID to CTD and to RNAP II could be demonstrated in vitro. Phosphorylation of a recombinant glutathione S-transferase-CTD by c-Abl was observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylated nor associated with the glutathione S-transferase-CTD in vivo. Transient overexpression of c-Abl also led to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II could be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II was again dependent on the presence of the CTD-ID in Abl. Finally, the ability of c-Abl to phosphorylate and associate with RNAP II could be correlated with the enhancement of transcription by c-Abl in transient cotransfection assays. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo.

Reflections on STAT3, STAT5, and STAT6 as fat STATs.

The current issue of the Proceedings contains an article entitled Defective STAT signaling by the leptin receptor in diabetic by Ghilardi et al. (1). The reported results suggest how leptin, the recently discovered weight control hormone (2), may signal cells through its cognate receptor by activation of STATs, proteins that serve the dual function of signal transducers and activators of transcription in cells exposed to signaling polypeptides (3, 4). Mice that produce no leptin (obese or ob mutants) weigh up to 60 g instead of the usual 15-20 g for a normal mouse. The human protein is virtually identical to mouse leptin, suggesting that the control of body weight in humans may also be regulated by this hormone (2). The effect of leptin on ob mice is to control food intake, so that weight loss ensues (5-7). In addition, the mice exhibit increased mouse-like exploratory activity. Thus, the description of the first molecule in the weight control pathway opens up the chance to explore in molecular detail the control of a complex behavior. The Ghilardi et al. paper (1) reports confirmatory results showing the presence in cells of widely scattered tissues, including the hypothalamus, the putative control center for feeding behavior, of a and leptin receptor (8-10). The leptin receptor has considerable sequence similarity to the gp130 transmembrane receptor chain that pairs as the signaling molecule with a number of other transmembrane proteins to constitute the receptor for many ligands including interleukin (IL)-6, ciliary neurotrophic factor, leukemiainhibitory factor (11, 12). The leptin receptor appears not to function normally in the mouse mutant termed diabetes (db) because of a base change in an intron that leads to a frequent aberrant splice choice; the resulting mRNA retains a translation stop codon producing a truncated protein lacking approximately 270 amino acids of the cytoplasmic domain of the transmembrane receptor (9). The omission of these amino acids was hypothesized to prevent intracellular signaling occasioned by leptin binding to its cell surface receptor. Based on the homology between the leptin receptor and the gp130 transmembrane protein (8-12), the pathway through which the leptin receptor seemed likely to signal is the recently recognized JAK/STAT pathway (3, 4). All of the known receptors that contain gp130 have JAK kinases (tyrosine kinases) bound to their intracellular tails (11). After ligand-mediated receptor assembly, the JAKs become phosphorylated on tyrosine and thereby activated as tyrosine kinases. The intracellular tail of one or more receptor chains is then phosphorylated on one or more tyrosine residues (13-15), offering binding sites to the Src homology 2 groups of latent cytoplasmic proteins called STATs (15). The attached STATs become phosphorylated on tyrosine by the activated Jak kinases. The STATs then dimerize, translocate to the nucleus, and participate in transcriptional regulation by binding to specific DNA sites. In the mutant db receptor both the putative STAT-binding sites and the JAK-binding sites are missing. There are six mouse and human STATs known at present (seven if the duplicated STAT5A and STAT5B genes are considered as two) and at least STATI, STAT3, and STAT5 exhibit differentially spliced forms. Over 30 different polypeptides have been recorded that cause STAT activation in various mammalian cells (3, 4, 11). The most potent activation of STATs through the gp130-containing receptors is of STAT3 (15-18) and by sequence comparison the wild-type leptin receptor has potential docking sites for STAT3 molecules. Ghilardi et al. (1) now report success in showing STAT activation dependent on the long form of the leptin receptor. In a comprehensive set of experiments they cotransfected COS cells with either the long or short versions of the leptin receptor together with, individually, each of six mouse or human STAT proteins. Leptin treatment of the cells transfected with the long but not the short receptor resulted in activation of DNAbinding complexes containing, individually, STAT3, STAT5, or STAT6; STATI, STAT2, and STAT4 were not detectably activated. It will now be crucial to show whether the same set of STATs is activated by leptin in the hypothalamus, which is hypothesized to be the center for weight control in the Coleman model (19). In this model the ob gene product, a circulating hormone, would operate by binding to a hypothalamic receptor, the db gene product, to regulate feeding.

Ligand-independent Activation of Transforming Growth Factor (TGF) β Signaling Pathways by Heteromeric Cytoplasmic Domains of TGF-β Receptors

Transforming growth factor beta (TGF-beta) transduces signals through two related serine/threonine kinase receptors, the type I and type II receptors, which have the ability to interact with each other. In the heteromeric complex, the type II receptor is the primary determinant of ligand binding and phosphorylates the cytoplasmic domain of the type I receptor. Using a chimeric receptor strategy, we and others have shown previously that a functional TGF-beta receptor complex requires heteromerization of both extracellular and intracellular domains of type I and type II receptors. In the current study, we show that overexpression of two receptors carrying a heteromeric combination of cytoplasmic domains resulted in ligand-independent responses, further supporting the functional requirement of the two heterologous cytoplasmic domains in TGF-beta signaling. Furthermore, coexpression of only the cytoplasmic domains of both the type I and II receptors or tethering the type II to the type I cytoplasmic domain activated TGF-beta responses in a ligand-independent manner. In cotransfected COS-1 cells, both cytoplasmic domains are associated with each other. Our results indicate that the cytoplasmic domains of the type I and type II TGF-beta receptors physically and functionally interact with each other in the heteromeric complex.

Examination of the Potential Functional Role of Conserved Cysteine Residues in the Hormone Binding Domain of the Human 1,25-Dihydroxyvitamin D3 Receptor

The significance of conserved cysteines at positions 288, 337, and 369 in the hormone binding domain of the human vitamin D receptor was evaluated by individual site-directed mutagenesis to glycine. Neither nuclear localization nor heterodimerization with retinoid X receptors in binding to the vitamin D-responsive element was appreciably affected by altering these cysteines, but vitamin D hormone (1,25-(OH)2D3) activated transcription was compromised significantly in the C288G and C337G mutants. Only the C288G mutant displayed depressed (3-fold) 1,25-(OH)2D3 ligand binding affinity at 4 degrees C, in vitro, although at elevated temperatures (23-37 degrees C), ligand binding was attenuated severely in C288G, moderately in C337G and very mildly in C369G. The degree of impairment of ligand binding at physiologic temperatures correlated with the requirement for increased concentrations of 1,25-(OH)2D3 ligand to maximally stimulate transcriptional activity in co-transfected COS-7 cells. Thus cysteine 288 and, to a lesser extent, cysteine 337 are important for high affinity hormone binding to the vitamin D receptor, which ultimately leads to ligand-dependent transcriptional activation.

Selective G Protein Coupling by C-C Chemokine Receptors

The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through G beta gamma released from the Gi proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the Gq class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C-X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the Gq class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the Gi-Gbetagamma-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs.

The functional role of the gamma-chain cysteine in Fc gamma RI-mediated phagocytosis using co-transfected COS-7 cells.

The functional role of the gamma-chain cysteine in Fc gamma RI-mediated phagocytosis using co-transfected COS-7 cells.

Mechanism of Interferon Action: Biochemical and Genetic Evidence for the Intermolecular Association of the RNA-Dependent Protein Kinase PKR from Human Cells

The interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation. Structure–function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1–551)K296R display a dominant negative behavior when expressed in transfected cells. The potential for PKR to form protein multimers has therefore been examined. Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA. First, the intermolecular association of PKRin vitrowas demonstrated in the context of an enzyme–substrate interaction. Purified recombinant histidine-tagged PKR(1–551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA. At a fixed RNA concentration, high concentrations of the HIS-PKR(1–551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself. Second, the yeast two-hybrid system was used to probe the intermolecular association of PKRin vivo.Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1–551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes,HIS3 andlacZ. The full-length RNA-binding deficient PKR(1–551)K64E/K296R double mutant also interacted with PKR(1–551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1–280)wt and PKR(281–551)K296R as well as p53, RAS, and BCL2 did not. Third, both PKR(1–551)K296R and PKR(1–551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells. By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1–551)K296R or PKR(1–551)K64E/K296R. These results indicate that PKR interacts with itself in an intermolecular manner bothin vivoandin vitro,and that RNA binding is neither necessary nor sufficient for PKR multimerization.

Epitope Tagging of the Human Endoplasmic Reticulum HSP70 Protein, BiP, to Facilitate Analysis of BiP-Substrate Interactions

We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitope-tag sequence close to the C-terminus (BiP-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antibody both immunoprecipitates BiP-tag and detects it on Western blots. Using transient expression of cDNAs in COS cells, we studied the interaction of BiP-tag with several membrane proteins. Consistent with previous work on BiP, BiP-tag bound poorly and transiently to newly made wild-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made erythropoietin receptor (EPO-R) polypeptides are retained in the ER and degraded there; we show here that, in cotransfected COS cells, newly made EPO-R is bound to BiP-tag prior to its degradation. Thus, by several criteria the BiP-tag molecule is fully functional in binding newly made proteins. Because it can be immunoprecipitated by a readily available antibody, it offers several advantages to the study of protein folding in the ER and the role of chaperones in this process.

Identification of a nef allele that causes lymphocyte activation and acute disease in Macaque monkeys

Residues 17 and 18 In nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRP-SGDLYERLLRARGETYGRLLGEVEDGYSOSP from residues 10–43. The YXXL motifs In this context match very well with consensus sequences for SH2 binding domains and are similar to ones present In nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting peripheral blood mononuclear cell cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pigtailed monkeys characterized by severe diarrhea, rash, and extensive lymphoid proliferation In the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with arc In cotransfected COS cells, and both 60 kDa arc and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype, and they provide evidence of a role for nef in signal transduction and cellular activation.

Identification of a Discrete Region of the G Protein γ Subunit Conferring Selectivity in βγ Complex Formation

The identification of multiple G protein beta and gamma subunit subtypes suggests a potential diversity of beta gamma heterodimers, which may contribute to the specificity of signal transduction between receptors and effectors. The assembly of beta and gamma subtypes is selective. For example, gamma 1 can assemble with beta 1 but not with beta 2, whereas gamma 2 assembles with both beta isoforms. To identify the structural features of the beta and gamma subunits governing selectivity in heterodimer assembly, a series of nonisoprenylated chimeras of gamma 1 and gamma 2 was constructed, and their interaction with beta 1 and beta 2 was assessed by their ability to direct beta expression to the cytosol in cotransfected COS cells. All of the gamma 1/gamma 2 chimeras were capable of interacting with beta 1 as judged by the cotransfection assay. Chimeras containing gamma 2 sequence near the middle of the molecule between two conserved sequence motifs were capable of interacting as well with beta 2. Among 12 divergent residues in this region, it was found that replacement of three consecutive amino acids in gamma 1, Glu-Glu-Phe (residues 38-40), with the three corresponding amino acids of gamma 2, Ala-Asp-Leu (residues 35-37), conferred the ability to assemble with beta 2. The reciprocal chimera containing Glu-Glu-Phe in the context of gamma 2 failed to assemble with beta 2. The last residue of this triplet is occupied by a leucine in all known mammalian gamma subunits except gamma 1 and appears to be a key determinant of the ability of a gamma subunit to assemble with beta 2. This locus maps to a region of predicted alpha-helical structure in the gamma subunit, likely to represent a point of physical contact with the beta subunit.

A 2a/D 2 receptor interactions are not observed in COS-7 cells transiently transfected with dopamine D 2 and adenosine A 2a receptor cDNA

The rat D 2 receptor and the dog A 2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D 2 receptors/mg protein and approx. 5 pmol/mg protein of the A 2a receptor as judged by binding experiments with [ 3H]raclopride [ or[ 3 H]-N- propyl-apomorphine (NPA)] and [ 3H]-CGS 21680, respectively. The high affinity K D values were 0.43 and 19 nM for D 2 and A 2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A 2b receptors known to be present on virtually every cell. The two A 2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A 2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D 2 receptors in the co-transfected COS-7 cells in contrast to the increased K H , K L and R H values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A 2a and D 2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of G s protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A 2a receptors and D 2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.

Microsomal triglyceride transfer protein, the abetalipoproteinemia gene product, mediates the secretion of apolipoprotein B-containing lipoproteins from heterologous cells.

Apolipoprotein (apo) B is an obligatory component of triglyceride-rich lipoproteins. In the rare autosomal recessive disorder abetalipoproteinemia (ABL), no triglyceride-rich lipoproteins are secreted. Mutations in the gene encoding the 97-kDa subunit of a microsomal triglyceride transfer protein (MTP) cause ABL (Sharp, D., Blinderman, L., Combs, K. A., Klenzle, B., Ricci, B., Wager-Smith, K., Gil, C. M., Turck, C. W., Bouma, M. E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature 365, 65-69; Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J. (1993) Hum. Mol. Genet. 2, 2109-2116). Here we have examined whether MTP is both necessary and sufficient to mediate the secretion of apoB-containing lipoproteins from cells that do not normally express either of these proteins. Carboxyl-terminal truncated forms of apoB, apoB17, and apoB41 on the centile system were expressed in COS-1 cells. ApoB17 was secreted whereas apoB41 was unable to traverse the secretory pathway. Cotransfection of apoB41 and MTP promoted the secretion of apoB41 as a buoyant lipoprotein particle with a modal density of 1.15 g/ml. When cotransfected COS-1 cells were cultured under conditions that increase the secretion of apoB100 from HepG2 cells, secretion of apoB41 was similarly increased. N-Acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain I inhibitor, abolished intracellular degradation of apoB41 and increased secretion 2.5-fold. Oleate, a substrate for triglyceride synthesis, reduced degradation from 50 to 19% and increased secretion by 2.5-fold. The effects of ALLN and oleate were additive. We conclude that the secretion of apoB from COS-1 cells cotransfected with apoB and MTP is determined by the competitive processes of lipoprotein assembly and intracellular degradation in the endoplasmic reticulum and that MTP is the only tissue-specific component, other than apoB, required for the secretion of apoB-containing lipoproteins.