Receptors & signal transduction最新文献

We have earlier reported that polyclonal antisera raised in rabbits to a luteinizing hormone/chorionic gonadotropin receptor (LH-R) purified from sheep luteal tissue has antibodies exhibiting hormone agonistic and antagonistic activities. Western blot analysis showed this antibody (LHR-anti IgG) to be highly specific to sheep luteal LH receptor (LH-R) (Jeyakumar and Moudgal, 1991). Using this, along with a battery of mouse monoclonal antibodies (MAbs) to hCG, an attempt has been made to better understand the interaction of LH/hCG with its receptor. Of the eight hCG MAbs screened, three (B14/B7, B52/18 and A7/G4) were specific to the beta-subunit; while a second set of three (G10/F7, H9/E9 and B52/21) were specific to the alpha-subunit. Two additional MAbs (B52/28 and F9/G8) did not recognize individual subunits, but bound like the rest intact hCG. Both 125I hCG and 125I anti LHR-IgG bound specifically to ovine luteal membrane LH-R. Assuming that a certain degree of similarity should exist between hCG and LHR-anti IgG, different hCG MAbs were tested for their ability to block the binding of either 125I hCG or 125I LHR-anti IgG to sheep luteal LH-R. It appears that hCG and LH-R share a minimum of four sites that are complementary to each other and these are recognized by the hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9. Whereas two of the MAbs B14/B7 and G10/F7 blocked the binding of both 125I labeled hCG and LHR-anti IgG to the receptor, MAbs A7/G4 and H9/E9 only inhibited the binding of 125I LHR-anti IgG to the LH-R. Although individually B14/B7 and G10/F7 blocked the binding of 125I LHR-anti IgG to LH-R to a maximum extent of 43%, together they inhibited binding by as much as 80%. The ability of B14/B7 to inhibit binding of 125I LHR-anti IgG to the receptor was also significantly increased by the addition of A7/G4. Finally, by demonstrating direct binding of the immobilized hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9 to LHR-anti IgG, we have been able to establish that the receptor binding sites of hCG and LHR-anti IgG are complementary and that a set of four sites are recognizable by the hCG MAbs. From the degree of interaction, it appears that two sites recognized by MAbs B14/B7 and G10/F7 (representing a site each in the beta- and alpha-subunit of hCG) have a prominent role in the interaction of hCG with its receptor. Thus, this study has provided us with an opportunity to investigate the interaction of LH/hCG with its receptor by an indirect approach of monitoring the binding of their respective antibodies with each other.

Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law (Km, 9.42 microM and Vmax, 77.7 nmol x min(-1) x 4 x 10(6) trypomastigotes(-1)). Preloaded [3H]ADPi was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote [ATP]o <--> ATP-R --> [ATP]i transport was [ATP]o-dependent and saturable at 100 microM. [ATP]o <--> ATP-R --> [ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o <--> ATP-R --> [ATP]i transport was also inhibited by the serine/threonine kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors and the inhibitor of tyrosine kinase growth factor receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [gamma32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (p92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.

The human mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily, which includes receptors for retinoic acid, vitamin D, and other steroids, such as the glucocorticoids (which bind the glucocorticoid receptor, GR). MR and GR, the corticosteroid receptors, share significant homology and are activated by steroid binding, resulting in a conformational change, nuclear translocation, and DNA binding. Despite these similarities with GR, the MR remains less well characterized. However, protein components known to be present in the unliganded GR are also likely to be components of the heteromeric MR complex. In the current study, we investigated whether or not hsp70, hsp90, and the immunophilin FKBP-52 are present in the nonsteroid-bound MR complex, because these proteins are known to be present in the unliganded GR complex. The unliganded MR complex was assembled in vitro using reticulocyte lysate and in vivo using the baculovirus overexpression system and Spodoptera frugiperda (Sf9) cells. Western blot analysis revealed the presence of hsp70, hsp90, and FKBP-52 in the unliganded complexes, but hsp90 and FKBP-52 were not detected following exposure to aldosterone. Electrophoretic mobility shift analysis demonstrated that DNA binding of MR occurred only after treatment with aldosterone. These studies indicate that proteins associated with the unliganded GR are also present in the unliganded MR complex, and that hsp90 and FKBP-52 dissociate prior to DNA binding in a manner similar to that described for GR. Finally, the stoichiometric analysis of the proteins present within the heteromeric MR complex suggests a divergence between this receptor and the GR.

The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

The p21-activated kinase (PAK) family includes protein phosphotransferases regulated by the GTPases rho, rac, and cdc42. Sequence homology, activation mechanism, and substrate specificity suggest that the well-characterized human placenta S6/H4 kinase is a member of this family. In these studies, S6/H4 kinase purified to homogeneity from human placenta was activated in vitro by cdc42-GTP, or protease incubation and MgATP-dependent autophosphorylation. The cdc42-activated enzyme demonstrated an Mr 60,000, and shares sequence homology with the gammaPAK family. Antipeptide antibodies against one of the autophosphorylation site sequences recognized a single p60 protein in the purified placenta preparation or Jurkat cell extracts. An autophosphorylated Mr 40,000 protein, previously identified as the catalytic domain of the enzyme, was also detected by the antibody after protease activation. Crude PAK60 obtained from Mono Q chromatography of Jurkat cell extracts and purified placenta enzyme catalyzed phosphorylation of histone H4 and myelin basic protein as well as a variety of synthetic peptides previously identified as S6/H4 kinase substrates. In addition, Jurkat myosin II and the regulatory myosin light chain were phosphorylated by the Jurkat and placenta gammaPAK. Synthetic peptides were used to demonstrate that the site of light chain phosphorylation occurs at the serine which results in ATPase activation. The data suggest that human gammaPAK may regulate cell motility by a GTP-dependent and calcium-independent mechanism.

Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveolar cells secrete surfactant in response to such secretagogs as terbutaline, calcium (Ca) ionophores (e.g., ionomycin [Io]), and adenosine triphosphate (ATP). A cell membrane receptor for SP-A, one of the surfactant proteins, regulates secretion by negative feedback. We used quantitative fluorescence microscopy to study the effects of SP-A on alterations in cytosolic Ca2+ ([Ca2+]i) elicited by surfactant secretagogs. Freshly isolated type II cells were loaded with Fura-2, then treated with secretagog, in the presence or absence of SP-A. Io and ATP produced biphasic increases in cytosol [Ca2+]i, reflecting first Ca2+ release from intracellular stores, and then influx through the cell membrane. Thapsigargin (TG) and Io directly initiate Ca2+ release; ATP elicits Ca2+ release via receptor-mediated mechanisms. Ca2+ release causes cell membrane Ca channels to open by as yet poorly understood mechanisms. Io itself acts as an additional Ca2+ channel. SP-A blocks much of the Ca2+ release and some of the Ca2+ influx elicited by these secretagogs. Antibody against SP-A receptor restores secretagog-induced Ca2+ fluxes from inhibition by SP-A, confirming that the inhibitory activity of SP-A is mediated through its receptor. Type II cells incubated in Ca2+-free medium plus SP-A show diminished Ca2+ release responses to TG or ATP, suggesting that the action of SP-A to prevent secretagog initiated increases in [Ca2+]i may reflect its ability to block Ca2+ release from cytoplasmic Ca stores. The feedback inhibition of surfactant secretion by SP-A may, correspondingly, be a manifestation of this effect. Because recent work suggests that TGF-beta also inhibits Ca2+ fluxes, SP-A and TGF-beta could be representative of a group of physiologic regulators that act by modulating intracellular Ca signaling.

In the usual formulation of the equations for competitive binding assays, the free concentrations of the various unlabeled ligands are approximated by the known values of their total concentrations, since the free concentrations are not easily determined. Although equations have been derived previously that give the exact solution with the free concentrations of unlabeled ligands treated as variables, these have not been useful in practice. We have devised a mathematical model for the competitive binding that which is both exact and practical for the general case of one labeled ligand, any number of unlabeled ligands, and any number of classes of binding sites. In this model, the total concentrations of unlabeled ligands are the explicit variables, instead of their free concentrations. The free concentrations of unlabeled ligands can be estimated from the model. The model is based on the law of mass action and the dilution principle, as well as a new concept, called the equivalent competitive binding principle.

Models of the m2 muscarinic receptor have been built and acetylcholine and an antagonist of the quinuclidinyl benzilate family docked to the putative active site. We have incorporated aspects of homology, site-directed mutagenesis studies and structure-activity studies of specific lead compounds in the construction of our receptor models with a primary focus on the structure of the binding sites. We have observed a deep pocket binding of 5-BrQNT, suggesting a plausible explanation for the observation that agonists and antagonists do not bind competitively. The results of these computational studies are interpreted within the context of the observed in vitro results. Our goal is to assist in the development of subtype receptor selective radiopharmaceuticals for use in PET and SPECT.

Rat prostate-cancer-cell stable-transfectants expressing either antisense-fibroblast growth factor (FGF-1) or antisense-FGF-2 transcripts that respectively have either undetectable FGF-1 or profoundly diminished FGF-2 protein content, were used for analyses of FGF-2 and/or 12-O-tetradecanoylphorbol 12-acetate (TPA) modulation of cell proliferation. Antisense-FGF-2 transfectant doubling-time was 2.6-fold greater than that of vector-control transfectants. FGF-2 and TPA respectively caused 2.5- and 3.0-fold reductions in antisense-FGF-2 transfectant doubling-time. Culture of antisense-FGF-2 transfectants in medium containing both FGF-2 and TPA further reduced their doubling time; however, this effect was not statistically different from that achieved by TPA treatment alone. Antisense-FGF-1 transfectant doubling-time was 2.2-fold greater than that of vector-control transfectants and was reduced 2.0- or 2.3-fold, respectively, when these cells were cultured in medium containing FGF-2 or TPA. In contrast to the results for antisense-FGF-2 transfectants, culture of antisense-FGF-1 transfectants in medium containing both FGF-2 and TPA caused a 2.6-fold reduction in transfectant doubling-time that was significantly greater than that caused by independent treatment with either FGF-2 or TPA. FGF-2 promoted rapid activation of rat prostate-cancer-cell PKCalpha and PKCepsilon, as assessed by isozyme translocation from the soluble to particulate cell fraction, and only moderately altered PKCdelta distribution. By contrast, TPA promoted rapid activation of all three PKC isozymes. Both the TPA- and FGF-2-mediated PKC activation were prolonged and possibly involved cyclic redistribution of isozymes between soluble and particulate cell fractions. FGF-2 also caused rapid phosphorylation of prostate-cancer-cell Shc, the adapter protein that mediates FGF-receptor-modulated ras signaling. The results of these studies indicate that FGF-2 and TPA independently and conjointly modulate rat prostate-cancer-cell antisense-transfectant doubling time and suggest that effector modulation of rat prostate-cancer-cell proliferation is achieved by processes involving PKC and/or ras mediated signaling.

The type I insulin-like growth factor receptor (IGF-IR) plays a crucial role in cell growth, transformation and protection from apoptosis. Although the mitogenic function of the IGF-IR may require the activation of insulin receptor substrate-1 (IRS-1) or IRS-2, an overexpressed IGF-IR is able to protect 32D cells, which lack IRS-1 and IRS-2, from apoptosis caused by Interleukin-3 (IL-3) withdrawal. Here, using mutational analysis, the authors identify domains of the IGF-IR necessary to protect from apoptosis without downstream signaling from IRS-1 and IRS-2. A receptor mutant of the tyrosine kinase (TK) domain only partially inhibited antiapoptotic signaling, whereas a mutant displaying constitutive autophosphorylation of the receptor did not show enhanced survival activity. Surprisingly, survival signaling was dependent upon tyrosine 950, the binding site for IRS-1, IRS-2, and Shc proteins. Yet, overexpressed Shc and/or IRS-1 could not replace the IGF-IR survival signal, suggesting the existence of other critical substrates. Finally, the C-terminus may encode a proapoptotic signal, as receptors truncated at C-terminal residues 1229 or 1245 were found to inhibit apoptosis better than the wild type (WT) IGF-IR.