Thyroid gangliosides with high affinity for thyrotropin: potential role in thyroid regulation

Thyroid cell membranes contain a multiplicity of gangliosides, some of which inhibit thyrotropin binding to thyroid membranes. The most potent inhibitor is a ganglioside which is present in only trace amounts and appears to have a novel structure. Thyroid gangliosides may play a role in relaying the hormonal message to the thyroid cell.     

Zinc mediation of the binding of human growth hormone to the human prolactin receptor

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.     

Gonadotropin-releasing hormone binding sites in human breast carcinoma

Gonadotropin-releasing hormone analogs can cause regression of hormone-dependent breast carcinomas. These effects are thought to be mediated through the inhibition of gonadotropic and steroid hormones. These analogs may also act directly on the tumor because they are effective in treating breast cancer in some postmenopausal women. The presence of specific binding sites for gonadotropin-releasing hormone was demonstrated in human breast carcinomas by means of a novel approach of ligand immunoblotting. The results indicate a possible mechanism by which the peptide has direct effects on this tissue. These binding proteins were not detectable in non-neoplastic breast tissue.     

Demonstration of a saturable binding site for thyrotropin in Yersinia enterocolitica

Several lines of evidence suggest that there might be immunologic cross-reactivity between the thyroid plasma membrane in humans and antigenic determinants in the enteric pathogen Yersinia enterocolitica. Studies were therefore performed to determine whether Y. enterocolitica, like the thyroid membrane, contains a thyrotropin binding site. A saturable binding site for bovine thyrotropin was indeed demonstrable, particularly in preparations of the organism that have been treated with ethylenediaminetetraacetate and lysozyme. Hormonal specificity of the binding site, as judged from the inhibition of binding of 125I-labeled bovine thyrotropin, was similar to that of the thyrotropin receptor in human thyroid tissue.     

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.     

Beta-adrenergic receptor: stereospecific interaction of iodinated beta-blocking agent with high affinity site

An iodine-labeled beta-adrenergic inhibitor ((125)l-hydroxybenzylpindolol) binds specifically to a site on turkey erythrocyte membranes. A series of beta-adrenergic agonists and inhibitors compete for this binding site, with apparent affinities paralleling biological effectiveness as activators or inhibitors of catecholaminestimulated adenylate cyclase. The activity of d-(+) agonists or inhibitors was 1 percent (or less) than that of the corresponding l-(-) isomers in competing for binding of the iodinated blocker as well as in affecting catecholamine-stimulated adenylate cyclase. 1-(-)-Norepinephrine was about one-tenth as active as l-(-)-isoproterenol in competing for the beta-blocking agent site. The stereospecificity of the interaction with the iodinated beta-blocking agent and the correspondence between affinity for site and biological potency of analogs suggested that this interaction is involved in function of the beta-adrenergic receptor.     

Adenosine 3',5'-monophosphate modulates thyrotropin receptor clustering and thyrotropin activity in culture

A biologically active rhodamine conjugate of thyrotropin binds at 4 degrees C to diffusely distributed membrane thyrotropin receptors which patch and become endocytosed into thyroid cells in a temperature-sensitive process. When the cells are first incubated with 8-bromo-cyclic adenosine monophosphate at 37 degrees C, the conjugate also binds to clustered receptors at 4 degrees C. Furthermore, 8-bromo-cyclic adenosine monophosphate reduces the amount of adenosine 3',5'-monophosphate (cyclic AMP) induced by thyrotropin. Hence, increased intracellular cyclic AMP induces receptor patching and reduces the concentration of cyclic AMP normally induced by thyrotropin. This suggests that cyclic AMP acts both as the second messenger of thyrotropin and also as the regulator of the level of thyrotropin receptors.     

An immunological investigation of human pituitary growth hormone

Growth hormone isolated from human pituitaries has been demonstrated to be a good antigen in the rabbit. With the rabbit antiserum to human somatotropin, it is possible to detect as little as 0.1 microg of the hormone by precipitin test. The antiserum was also capable of neutralizing the growth-promoting activity of human somatotropin.     

Quantitative analysis of D2 dopamine receptor binding in the living human brain by PET

D2 dopamine receptors in the putamen of living human subjects were characterized by using the selective, high-affinity D2 dopamine receptor antagonist carbon-11-labeled raclopride and positron emission tomography. Experiments in four healthy men demonstrated saturability of [11C]raclopride binding to an apparently homogeneous population of sites with Hill coefficients close to unity. In the normal putamen, maximum binding ranged from 12 to 17 picomoles per cubic centimeter and dissociation constants from 3.4 to 4.7 nanomolar. Maximum binding for human putamen at autopsy was 15 picomoles per cubic centimeter. Studies of [11C]raclopride binding indicate that clinically effective doses of chemically distinct neuroleptic drugs result in 85 to 90 percent occupancy of D2 dopamine receptors in the putamen of schizophrenic patients.     

Transition state analogs as ligands for affinity purification of juvenile hormone esterase

Insect juvenile hormones are metabolized in numerous species of caterpillars by low abundance, highly specific esterases. Because of their role in regulating and possibly disrupting juvenile hormone titer and thus insect metamorphosis, they are of interest to developmental biologists as well as scientists interested in selective insect control. However, the enzymes have defied attempts to purify and characterize them. Juvenile hormone esterase activity can be inhibited by a variety of 3-substituted 1,1,1-trifluoropropanone sulfides. These apparent transition state analogs were used as ligands and eluting agents to purify juvenile hormone esterase from four insect species from 500-fold to over 1000-fold in high yield. After elution from the affinity column, the enzymes were radiolabeled with paraoxon and analyzed by electrophoresis, and the results demonstrate a high degree of purity. Transition state analogs may be useful for the affinity purification of other enzymes.     

An estrogen-binding protein and endogenous ligand in Saccharomyces cerevisiae: possible hormone receptor system

A protein macromolecule in the cytosol of the unicellular eukaryotic yeast Saccharomyces cerevisiae selectively binds the vertebrate estrogen hormone 17 beta-estradiol with high affinity. Lipid extracts of the yeast cells or the conditioned growth medium yield a substance that can bind competitively to the tritiated estradiol-binding sites in the yeast and to mammalian estrogen receptors. These findings suggest that the binding protein may be a primitive hormone receptor and that the lipid-extractable substance represents the endogenous ligand.     

An LRR receptor kinase involved in perception of a peptide plant hormone,phytosulfokine

The sulfated peptide phytosulfokine (PSK) is an intercellular signal that plays a key role in cellular dedifferentiation and proliferation in plants. Using ligand-based affinity chromatography, we purified a 120-kilodalton membrane protein, specifically interacting with PSK, from carrot microsomal fractions. The corresponding complementary DNA encodes a 1021-amino acid receptor kinase that contains extracellular leucine-rich repeats, a single transmembrane domain, and a cytoplasmic kinase domain. Overexpression of this receptor kinase in carrot cells caused enhanced callus growth in response to PSK and a substantial increase in the number of tritium-labeled PSK binding sites, suggesting that PSK and this receptor kinase act as a ligand-receptor pair.     

A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid

The plant hormone abscisic acid (ABA) regulates many physiological and developmental processes in plants. The mechanism of ABA perception at the cell surface is not understood. Here, we report that a G protein-coupled receptor genetically and physically interacts with the G protein alpha subunit GPA1 to mediate all known ABA responses in Arabidopsis. Overexpressing this receptor results in an ABA-hypersensitive phenotype. This receptor binds ABA with high affinity at physiological concentration with expected kinetics and stereospecificity. The binding of ABA to the receptor leads to the dissociation of the receptor-GPA1 complex in yeast. Our results demonstrate that this G protein-coupled receptor is a plasma membrane ABA receptor.     

Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR

Binding of virus particles to specific host cell surface receptors is known to be an obligatory step in infection even though the molecular basis for these interactions is not well characterized. The crystal structure of the adenovirus fiber knob domain in complex with domain I of its human cellular receptor, coxsackie and adenovirus receptor (CAR), is presented here. Surface-exposed loops on knob contact one face of CAR, forming a high-affinity complex. Topology mismatches between interacting surfaces create interfacial solvent-filled cavities and channels that may be targets for antiviral drug therapy. The structure identifies key determinants of binding specificity, which may suggest ways to modify the tropism of adenovirus-based gene therapy vectors.