Mechanism of interleukin-2 signaling: mediation of different outcomes by a single receptor and transduction pathway

The T cell lymphokine, interleukin-2 (IL-2), plays a pivotal role in an immune response by stimulating antigen-activated B lymphocytes to progress through the cell cycle and to differentiate into antibody-secreting cells. An IL-2 inducible B lymphoma line, in which the growth and differentiation responses are uncoupled, provides a model system for dissecting the signaling mechanisms operating in each response. This system was used to show that both signals are initiated by IL-2 binding to a single, unifunctional receptor complex. Moreover, both signals are transduced by a pathway that does not involve any known second messenger system and that can be blocked by a second T cell lymphokine, interleukin 4. These findings suggest that the pleiotrophic effects of IL-2 are determined by different translations of the signal in the nucleus.     

Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane

Phototransduction in rod cells is likely to involve an intracellular messenger system that links the absorption of light by rhodopsin to a change in membrane conductance. The direct effect of guanosine 3',5'-monophosphate (cGMP) on excised patches of rod outer segment membrane strongly supports a role for cGMP as an intracellular messenger in phototransduction. It is reported here that magnesium and calcium directly affect the conductance of excised patches of rod membrane in the absence of cGMP and that magnesium, applied to intact rod cells, blocks a component of the cellular light response. The divalent cation-suppressed conductance in excised patches showed outward rectification and cation-selective permeability resembling those of the light-suppressed conductance measured from the intact rod cell. The divalent cation-suppressed conductance was partly blocked by a concentration of the pharmacological agent L-cis-diltiazem that blocked all of the cGMP-activated conductance. Divalent cations may act, together with cGMP, as an intracellular messenger system that mediates the light response of the rod photoreceptor cell.     

Differences in adrenergic recognition by pancreatic A and B cells

The adrenergic control of glucose homeostasis is mediated in part through variations in the release of pancreatic hormones. In this study, purified pancreatic A and B cells were used to identify the recognition and messenger units involved in the adrenergic regulation of glucagon and insulin release. Catecholamines induced beta-adrenergic receptor activity in A cells and alpha 2-adrenergic receptor activity in B cells. The two recognition units provoked opposite variations in the production of cellular cyclic adenosine monophosphate, the beta-adrenergic unit enhancing the nucleotide's permissive effect on amino acid-induced glucagon release and the alpha 2-adrenergic unit inhibiting that upon glucose-induced insulin release. In both cell types, catecholamines interact powerfully with the synergistic control of hormone release by nutrient- and (neuro)hormone-driven messenger systems.     

Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice

Three lines of transgenic mice were produced that develop pancreatic neoplasms as a consequence of expression of an elastase I-SV40 T-antigen fusion gene in the acinar cells. A developmental analysis suggests at least a two-stage process in the ontogeny of this disease. The first stage is a T antigen-induced, preneoplastic state characterized by a progression from hyperplasia to dysplasia of the exocrine pancreas, by an increased percentage of tetraploid cells, and by an arrest in acinar cell differentiation. The second stage is characterized by the formation of tumor nodules that appear to be monoclonal, because they have discrete aneuploid DNA contents. The cells within the nodules as compared to normal pancreatic tissue have less total RNA by a factor of 5, less pancreas-specific messenger RNA by a factor of about 50, and increased levels of T-antigen messenger RNA. A tumor cell line has been derived that retains both pancreatic and neoplastic properties.     

A spatial-temporal model of cell activation

A spatial-temporal model of calcium messenger function is proposed to account for sustained cellular responses to sustained stimuli, as well as for the persistent enhancement of cell responsiveness after removal of a stimulus, that is, cellular memory. According to this model, spatial separation of calcium function contributes to temporal separation of distinct phases of the cellular response. At different cellular sites, within successive temporal domains, the calcium messenger is generated by different mechanisms and has distinct molecular targets. In particular, prolonged cell activation is brought about by the interaction of calcium with another spatially confined messenger, diacylglycerol, to cause the association of protein kinase C with the plasma membrane. Activity of the membrane-associated protein kinase C is controlled by the rate of calcium cycling across the plasma membrane. In some instances, a single stimulus induces both protein kinase C activation and calcium cycling and thus causes prolonged activation; but in others, a close temporal association of distinct stimuli brings about cell activation via interaction of these intracellular messengers. Persistent enhancement of cell responsiveness after removal of stimuli is suggested to be due to the continued association, or anchoring, of protein kinase C to the membrane.     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.     

Serological visualization of interleukin 2

Interleukin 2, a lymphokine that acts as a second signal of cellular immune response by way of its action as a T-cell growth factor, was morphologically identified by immunoperoxidase staining. With the use of a monoclonal antibody to interleukin 2 and several complex-forming antisera, the lymphokine was readily distinguished in cytocentrifuge preparations of peripheral blood leukocytes stimulated with a T-cell mitogen. When preparations of cloned interleukin 2 producer and responder cells were stained by the same procedures, discrete patterns of both responder and producer cell phenotypes were revealed. Interleukin 2 producer T cells exhibited a characteristic intense, ringlike cytoplasmic staining, whereas the responder cells (as exemplified by interleukin 2-dependent cell lines) exhibited a less intensive, spotlike membrane staining. In addition, intense membrane localization of interleukin 2, reminiscent of potential capping phenomena, could be observed in stained preparations of cloned responder cells.     

Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity

The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.     

Heterologous expression of excitability proteins: route to more specific drugs?

Many clinically important drugs act on the intrinsic membrane proteins (ion channels, receptors, and ion pumps) that control cell excitability. A major goal of pharmacology has been to develop drugs that are more specific for a particular subtype of excitability molecule. DNA cloning has revealed that many excitability proteins are encoded by multigene families and that the diversity of previously recognized pharmacological subtypes is matched, and probably surpassed, by the diversity of messenger RNAs that encode excitability molecules. In general, the diverse subtypes retain their properties when the excitability proteins are expressed in foreign cells such as oocytes and mammalian cell lines. Such heterologous expression may therefore become a tool for testing drugs against specific subtypes. In a systematic research program to exploit this possibility, major considerations include alternative processing of messenger RNA for excitability proteins, coupling to second-messenger systems, and expression of enough protein to provide material for structural studies.     

Identification of a thyroid hormone receptor that is pituitary-specific

Three cellular homologs of the v-erbA oncogene were previously identified in the rat; two of them encode high affinity receptors for the thyroid hormone triiodothyronine (T3). A rat complementary DNA clone encoding a T3 receptor form of the ErbA protein, called r-ErbA beta-2, was isolated. The r-ErbA beta-2 protein differs at its amino terminus from the previously described rat protein encoded by c-erbA beta and referred to as r-ErbA beta-1. Unlike the other members of the c-erbA proto-oncogene family, which have a wide tissue distribution, r-erbA beta-2 appears to be expressed only in the anterior pituitary gland. In addition, thyroid hormone downregulates r-erbA beta-2 messenger RNA but not r-erbA beta-1 messenger RNA in a pituitary tumor-derived cell line. The presence of a pituitary-specific form of the thyroid hormone receptor that may be selectively regulated by thyroid hormone could be important for the differential regulation of gene expression by T3 in the pituitary gland.     

Decoding in the absence of a codon by tmRNA and SmpB in the ribosome

In bacteria, ribosomes stalled at the end of truncated messages are rescued by transfer-messenger RNA (tmRNA), a bifunctional molecule that acts as both a transfer RNA (tRNA) and a messenger RNA (mRNA), and SmpB, a small protein that works in concert with tmRNA. Here, we present the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome at 3.2 angstroms resolution. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome and explains why the tmRNA-SmpB system does not interfere with normal translation.     

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.     

Left-right dynein motor implicated in selective chromatid segregation in mouse cells

During cell division, copies of mouse chromosome 7 are segregated selectively or randomly to daughter cells depending on the cell type. The mechanism for differential segregation is unknown. Because mouse left-right dynein (LRD) gene mutations result in randomization of visceral organs' laterality, we hypothesized that LRD may also function in selective chromatid segregation. Indeed, upon knock-down by RNA interference methods, LRD depletion disrupts biased segregation. LRD messenger RNA presence or absence correlates with the observed segregation patterns. This work supports the claim that LRD functions in a mechanism for selective chromatid segregation.     

Secretion of activin by interstitial cells in the testis

Activin, a dimer formed by the beta subunits of inhibin, has an effect that is opposite to that of inhibin in a number of biological systems. Which cell types secrete activin in vivo is not known. TM3 cells, a Leydig-derived cell line, contained messenger RNAs that hybridized with human beta A and beta B complementary DNA probes and were similar in size to the porcine messenger RNA for the beta subunits of inhibin. No hybridization to the inhibin alpha subunit was detectable in the TM3 cells. Conditioned medium from TM3 cells and from primary cultures of rat and porcine interstitial cells stimulated the release of follicle-stimulating hormone in a pituitary cell culture assay. It is likely that, in the testis, the Leydig cells secrete activin and the Sertoli cells produce inhibin, or a combination of both.     

S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth

The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth.