Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells

The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.     

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.     

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

cDNA cloning and expression of the peptide-binding beta subunit of rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1

Protein farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to cysteine in ras proteins and other membrane-associated proteins. The beta subunit contains the recognition site for the peptide substrates, but is inactive in the absence of the alpha subunit. A cloned cDNA for the rat beta subunit predicts a protein of 437 amino acids whose mRNA is present in many tissues. Transfection of the beta subunit cDNA produced farnesyltransferase activity in human kidney cells, but only when it was transfected together with a cDNA encoding part of the alpha subunit. Each of the subunits appeared to be unstable in the transfected cells unless the other subunit was present. The rat beta subunit shows 37% sequence identity with the protein encoded by the yeast DPR1/RAM1 gene, indicating that DPR1/RAM1 is the yeast counterpart of the peptide-binding subunit of the mammalian farnesyltransferase.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance

The phenotypic effects of selectively altering the levels of alpha B- crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B- crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

Murine recombinant Fc gamma RIII, but not Fc gamma RII, trigger serotonin release in rat basophilic leukemia cells.

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.     

Y receptor-mediated induction of CD63 transcripts, a tetraspanin determined to be necessary for differentiation of the intestinal epithelial cell line, hBRIE 380i cells.

Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY- and NPY-induced rearrangement of actin filaments may be in part through a Y1alpha and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with beta(1)-integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins.     

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation

Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.     

Immunodetection of alpha1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney.

The calcium channel alpha1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of alpha1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac alpha1E was determined by SDS-PAGE and immunoblotting (218 +/- 6 kD; n = 3). Compared to alpha1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of alpha1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of alpha1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of alpha1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of alpha1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca(2+)-dependent secretion of peptide hormones such as ANP and urodilatin.     

Differential degradation rates of the G protein alpha o in cultured cardiac and pituitary cells

Signal transduction in biological membranes is modulated by a family of GTP-binding proteins termed G proteins. Differences in the tissue-specific expression of G protein subtypes suggest that the levels of individual G proteins may be an important determinant of the hormonal response in a given cell type. We have used a polyclonal antibody raised against the purified G protein, alpha o to study alpha o in the rat pituitary cell line GH4 and in primary rat cardiocytes in culture by quantitative immunoprecipitation. Biosynthetic labeling and specific immunoprecipitation of alpha o in pulse-chase experiments demonstrated that the t1/2 for alpha o degradation is 28 +/- 7 h (n = 4) in GH4 pituitary cells and is greater than 72 h (n = 4) in cardiocytes. The steady-state level of alpha o protein is similar in both cell types as measured by Western blots. Northern blots of poly(A)-selected mRNA from these two cell types were probed with labeled alpha o cDNA and showed they have similar alpha o mRNA levels. The observation of different degradation rates, but similar steady-state protein levels, suggests that the rate of alpha o synthesis is different in GH4 cells and cardiocytes. Since mRNA levels are approximately equal in both, our studies imply that protein translation controls may be important determinants of G protein alpha subunit concentrations in biological membranes.     

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.     

Inhibition of glucocorticoid-induced apoptosis by the expression of antisense gene of mitochondrial ATPase subunit 6(1)

To isolate the apoptosis-linked genes involved in the cell death of thymocytes induced by glucocorticoids, we developed a functional cloning assay. Murine CD4(+)CD8(+) thymic cell line 2-257-20 cells were transfected with cDNA expression libraries obtained from a dexamethasone-resistant cell line. The transfected cells were selected in the presence of dexamethasone, and the plasmids which episomally expanded were then extracted from the surviving cells. One of the rescued cDNAs was found to be an antisense cDNA fragment identical to the mouse mitochondrial ATPase 6 gene. In the stable transfectants with the ATPase 6 antisense gene, the induction of apoptosis by dexamethasone was significantly delayed. Furthermore, the ATP synthesis in these transfectants was also reduced to some extent. ATPase 6 is a subunit of F(o)F(1) ATPase and our results support that ATP synthesis from the mitochondria is necessary for the induction of apoptosis induced by glucocorticoids.