Evidence that androgens are the primary steroids produced by Xenopus laevis ovaries and may signal through the classical androgen receptor to promote oocyte maturation.

Steroid-induced maturation of Xenopus oocytes has long served as a model for studying meiosis. Progesterone has been considered the relevant steroid controlling maturation, perhaps through interactions with classical progesterone receptors. In this study, we provide evidence that androgens, rather than progesterone, are the physiologic mediators of Xenopus oocyte maturation. Androgens were equal or more potent activators of maturation in vitro relative to progesterone and were significantly more abundant in the serum and ovaries of beta-human chorionic growth hormone-stimulated frogs. Androgen action appeared to be mediated by classical androgen receptors (ARs) expressed in oocytes, as androgen-induced maturation and signaling was specifically attenuated by AR antagonists. Interestingly, we found that progesterone was rapidly converted to the androgen androstenedione in isolated oocytes by the enzyme CYP17, suggesting that androgens may be promoting maturation even under conditions typical for "progesterone-mediated" maturation assays. Androgens are thought to play an important role in ovarian development as well as pathology, and signaling through the AR may prove to be a major regulatory mechanism mediating these processes.

G protein beta gamma subunits inhibit nongenomic progesterone-induced signaling and maturation in Xenopus laevis oocytes. Evidence for a release of inhibition mechanism for cell cycle progression.

Progesterone-induced maturation of Xenopus oocytes is a well known example of nongenomic signaling by steroids; however, little is known about the early signaling events involved in this process. Previous work has suggested that G proteins and G protein-coupled receptors may be involved in progesterone-mediated oocyte maturation as well as in other nongenomic steroid-induced signaling events. To investigate the role of G proteins in nongenomic signaling by progesterone, the effects of modulating Galpha and Gbetagamma levels in Xenopus oocytes on progesterone-induced signaling and maturation were examined. Our results demonstrate that Gbetagamma subunits, rather than Galpha, are the principal mediators of progesterone action in this system. We show that overexpression of Gbetagamma inhibits both progesterone-induced maturation and activation of the MAPK pathway, whereas sequestration of endogenous Gbetagamma subunits enhances progesterone-mediated signaling and maturation. These data are consistent with a model whereby endogenous free Xenopus Gbetagamma subunits constitutively inhibit oocyte maturation. Progesterone may induce maturation by antagonizing this inhibition and therefore allowing cell cycle progression to occur. These studies offer new insight into the early signaling events mediated by progesterone and may be useful in characterizing and identifying the membrane progesterone receptor in oocytes.

Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.

The thrombin receptor PAR1 becomes rapidly phosphorylated upon activation by either thrombin or exogenous SFLLRN agonist peptide. Substitution of alanine for all serine and threonine residues in the receptor's cytoplasmic carboxyl-terminal tail ablated phosphorylation and yielded a receptor defective in both shutoff and agonist-triggered internalization. These observations suggested that activation-dependent phosphorylation of PAR1's cytoplasmic tail is required for both shutoff and agonist-triggered internalization. To identify the phosphorylation site(s) that are necessary for these functions, we generated three mutant receptors in which alanine was substituted for serine and threonine residues in the amino-terminal, middle, and carboxyl-terminal thirds of PAR1's cytoplasmic tail. When stably expressed in fibroblasts, all three mutated receptors were rapidly phosphorylated in response to agonist, while a mutant in which all serines and threonines in the cytoplasmic tail were converted to alanines was not. This result suggests that phosphorylation can occur at multiple sites in PAR1's cytoplasmic tail. Alanine substitutions in the N-terminal and C-terminal portions of the tail had no effect on either receptor shutoff or agonist-triggered internalization. By contrast, alanine substitutions in the "middle" serine cluster between Ser(391) and Ser(406) yielded a receptor with considerably slower shutoff of signaling after thrombin activation than the wild type. Surprisingly, this same mutant was indistinguishable from the wild type in agonist-triggered internalization and degradation. Overexpression of G protein-coupled receptor kinase 2 (GRK2) and GRK3 "suppressed" the shutoff defect of the S --> A (391-406) mutant, consistent with this defect being due to altered receptor phosphorylation. These results suggest that specific phosphorylation sites are required for rapid receptor shutoff, but phosphorylation at multiple alternative sites is sufficient for agonist-triggered internalization. The observation that internalization and acute shutoff were dissociated by mutation of PAR1 suggests that there are quantitative or qualitative differences in the requirements or mechanisms for these two processes.

Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand.

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.

Termination of signaling by protease-activated receptor-1 is linked to lysosomal sorting.

The irreversible proteolytic mechanism by which protease-activated receptor-1 (PAR1), the G protein-coupled receptor (GPCR) for thrombin, is activated raises the question of how it is shut off. Like classic GPCRs, activated PAR1 is rapidly phosphorylated and internalized, but unlike classic GPCRs, which recycle, internalized PAR1 is sorted to lysosomes. A chimeric PAR1 bearing the substance P receptor's cytoplasmic carboxyl tail sequestered and recycled like wild-type substance P receptor. In cells expressing this chimera, signaling in response to the PAR1-activating peptide SFLLRN ceased as expected upon removal of this agonist. Strikingly, however, when the chimera was activated proteolytically by thrombin, signaling persisted even after thrombin was removed. This persistent signaling was apparently due to "resignaling" by previously activated receptors that had internalized and recycled back to the cell surface. Thus the cytoplasmic carboxyl tail of PAR1 specifies an intracellular sorting pattern that is linked to its signaling properties. In striking contrast to most GPCRs, sorting of activated PAR1 to lysosomes rather than recycling is critical for terminating PAR1 signaling-a trafficking solution to a signaling problem.

Gene and locus structure and chromosomal localization of the protease-activated receptor gene family.

Protease-activate receptors (PARs) mediate activation of platelets and other cells by thrombin and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis, and other normal and pathological processes. We report here the structure of the mouse and human PAR3 genes as well as the organization of a PAR gene cluster encompassing the genes encoding PARs 1, 2, and 3. We also report the structure of the mouse and human PAR4 genes, which map to distinct chromosomal locations and encode a new thrombin receptor. PARs 1-4 are all encoded by genes with the same two exon structure. In each case, exon 1 encodes a signal peptide, and exon 2 encodes the mature receptor protein. These are separated by an intron of variable size. The genes encoding PARs 1-3 all map to chromosome 13D2 in mouse and chromosome 5q13 in human. In mouse, all three genes are located within 80 kilobases of each other. The PAR1 gene is located centrally and is flanked upstream by the PAR3 gene and downstream by the PAR2 gene in both species. The proximity of the PAR1 and PAR3 genes suggests the possibility that these genes might share regulatory elements. A comparison of the structures of the PAR amino acid sequences, gene structures, locus organization, and chromosomal locations suggests a working model for PAR gene evolution.

Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3.

Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I.

Multiple arginine residues within the basic domain of HTLV-I Rex are required for specific RNA binding and function.

The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for viral replication, acting post-transcriptionally to enhance the expression of unspliced and singly spliced viral mRNAs that encode the Gag, Pol, and Env virion proteins. Rex function involves its direct interaction with a complex stem-loop structure termed the Rex RNA response element (RexRE), which is located within the 3' retroviral long terminal repeat. Binding of Rex to the RexRE involves a positively charged arginine-rich domain located near the N-terminus which also functions as a nuclear localization signal. Strikingly, substitution of all seven of the arginine residues present within this domain with positively charged lysine residues exerted no adverse effect on the nuclear targeting of Rex. However, these lysine substitutions completely abrogated both Rex binding to the RexRE and Rex function. Reversion of multiple substituted lysines to arginines at specific locations within this domain was required to restore both RexRE binding and biological function to the Rex protein. Thus, while the presence of positive charge alone in this domain appears sufficient for nuclear localization of Rex, multiple arginine residues at specific sites are essential for the full expression of RNA binding and functional activity of this retroviral trans-regulatory protein.

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor.

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.

Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor.

The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding sequence (pNEF-fs). Consistent with prior studies, the Nef proteins produced by pNEF and pNEF-2/3 were approximately 27 kDa in size, posttranslationally modified by myristoylation, and primarily associated with cytoplasmic membrane structures. However, in contrast to previous reports, these Nef proteins failed to inhibit transcriptional activity of the HIV-1 long terminal repeat in any of a variety of cell types, including primary human T lymphocytes, Jurkat or YT-1 leukemic T cells, U-937 promonocytic cells, and nonlymphoid COS cells. Furthermore, HXB-3 proviral clones of HIV-1 containing either a wild-type or mutated version of the nef gene replicated in an indistinguishable manner when transfected into COS cells. Our findings suggest that Nef is neither a transcriptional inhibitor nor a negative viral factor under these assay conditions. Rather, we suggest that the primary biological function of this conserved HIV-1 protein has yet to be defined, perhaps reflecting an intrinsic shortcoming in the in vitro experimental systems presently available for the study of HIV-1.

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation.

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.