Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.     

Creating a protein-based element of inheritance

Proteins capable of self-perpetuating changes in conformation and function (known as prions) can serve as genetic elements. To test whether novel prions could be created by recombinant methods, a yeast prion determinant was fused to the rat glucocorticoid receptor. The fusion protein existed in different heritable functional states, switched between states at a low spontaneous rate, and could be induced to switch by experimental manipulations. The complete change in phenotype achieved by transferring a prion determinant from one protein to another confirms the protein-only nature of prion inheritance and establishes a mechanism for engineering heritable changes in phenotype that should be broadly applicable.     

Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.     

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.     

猪卵泡抑制素受体的分离与纯化

通过分离纯化卵泡抑制素受体,确定其受体类型,旨在为抑制素的作用机制研究奠定基础.采集30头长白猪垂体组织制备膜蛋白,用亲和层析法从该蛋白组分中分离、纯化出抑制素受体.SDS-PAGE结果发现该受体提取物中有6种蛋白,分子质量从14 ku到130 ku不等,表明抑制素受体可能不是特异性的单一受体,而是共受体.改良的EIASA结果证实,该受体提取物特异性活性为99 U/mg,能与抑制素结合,且与剂量呈正相关(R=0.993).     

Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis

S-49 mouse lymphoma cells undergo lysis when treated with glucocorticoids; the mechanism of this effect is not understood. A protein was detected in the plasma membrane of these cells by means of direct immunofluorescent labeling with a monoclonal antibody to the soluble glucocorticoid receptor. Cellular heterogeneity in the content of this glucocorticoid receptor-like molecule was evident. By immunoadsorption to antibody-coated tissue culture plates, the cells were separated into populations positive (100%) and depleted (38%) for this membrane antigen. Gel electrophoresis, specific immunoblot, and autoradiographic (binding of [3H]dexamethasone mesylate) analysis of the membrane proteins from the membrane antigen-positive group revealed multiple protein bands ranging in size from 85 to 145 kilodaltons. Furthermore, comparison of the glucocorticoid sensitivity of these groups of cells showed complete lysis of the membrane antigen-positive cells and only partial lysis of the antigen-deficient group, which suggests that the lysis response of cells to glucocorticoids is mediated by a glucocorticoid receptor-like molecule located in the plasma membrane.     

Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system

Long-term exposure of various cell types to beta-adrenergic agonists such as isoproterenol leads to an attenuated responsiveness ("desensitization") of the adenylate cyclase system to further challenge with these agonists. The turkey erythrocyte model system was used earlier to show that a covalent modification of the receptor (phosphorylation) is associated with this process. The functionality of the "desensitized" beta-adrenergic receptor was assessed by implanting purified beta-adrenergic receptor preparations from control and desensitized turkey erythrocytes into phospholipid mixtures and then fusing them with receptor-deficient cells (Xenopus laevis erythrocytes). Desensitized beta-adrenergic receptors showed a 40 to 50 percent reduction in their ability to couple to the heterologous adenylate cyclase system, comparable to the reduction in their functionality observed in their original membrane environment. These results demonstrate the utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors and show a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system.