16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene.

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.

Pentameric subunit stoichiometry of a neuronal glutamate receptor.

Ionotropic glutamate receptors, neurotransmitter-activated ion channels that mediate excitatory synaptic transmission in the central nervous system, are oligomeric membrane proteins of unknown subunit stoichiometry. To determine the subunit stoichiometry we have used a functional assay based on the blockade of two alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunit 1 (GluR1) mutant subunits selectively engineered to exhibit differential sensitivity to the open channel blockers phencyclidine and dizolcipine (MK-801). Coinjection into amphibian oocytes of weakly sensitive with highly sensitive subunit complementary RNAs produces functional heteromeric channels with mixed blocker sensitivities. Increasing the fraction of the highly sensitive subunit augmented the proportion of drug-sensitive receptors. Analysis of the data using a model based on random aggregation of receptor subunits allowed us to determine a pentameric stoichiometry for GluR1. This finding supports the view that a pentameric subunit organization underlies the structure of the neuronal ionotropic glutamate receptor gene family.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld).