Ligand structure influences autologous downregulation of estrogen receptor-alpha messenger RNA.

A series of A- and D-ring substituted estrogen analogues have been examined for their effect on estrogen receptor-alpha (ERalpha) mRNA downregulation. Recently it has been proposed that ERalpha autologous downregulation occurs via transcriptional repression exerted by the binding of the ERalpha-ligand complex to the 5' region of the coding region of the ERalpha gene. Placement of the phenolic hydroxyl group on the various carbons of the aromatic A-ring of estratrien-17betaol (carbons 1-3) produced ligands which diminished the steady state level of ERalpha mRNA in relation to their affinity for receptor.

Signaling molecules involved in coupling growth hormone receptor to mitogen-activated protein kinase activation.

We have shown previously that GH stimulates the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal-regulated kinases) 1 and 2. To examine pathways coupling GH receptor (GHR) to MAP kinase activation, we have determined the effects of GH on SHC-growth factor receptor bound 2-son of Sevenless (SHC-Grb2-SOS) association and activation of Ras, Raf, and MAP-ERK kinase (MEK). GH promoted the rapid, transient association of SHC with the Grb2-SOS complex, which correlated with the time course of Ras, Raf, and MEK activation.

Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B.

GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B.

Cholecystokinin stimulates formation of shc-grb2 complex in rat pancreatic acinar cells through a protein kinase C-dependent mechanism.

Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains.

Signalling pathway of GH.

GH has long been known as a regulator of body growth and metabolism, yet its mechanism of action at the cellular level has been elusive. We have recently shown that GH promotes the rapid association of GH receptor with the tyrosine kinase JAK2, activates JAK2, and promotes the tyrosyl phosphorylation of both JAK2 and GH receptor. This suggests that the initial signalling event in GH action is the activation of JAK2 which in turn phosphorylates tyrosines within JAK2 and GH receptor.

Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor.

Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine kinase activity, GH has recently been shown to promote the association of its receptor with JAK2 tyrosine kinase, to activate JAK2, and to promote the tyrosyl phosphorylation of both GH receptor (GHR) and JAK2. In this work, we examined whether tyrosines 333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH.

Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC association with Grb2.

Growth hormone (GH) has been shown to stimulate the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2 are activated by tyrosine kinases involves the Src homology (SH)-2 containing proteins SHC and Grb2. To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH.

Effect of A-ring isomers of estradiol-17 beta on gene products in MCF-7 cells.

Two-dimensional polyacrylamide gel-electrophoresis analysis of in vitro translation products from extracted cellular mRNAs was utilized to examine the effect of A-ring isomers of estradiol (E2) on the synthesis of proteins involved in the response of MCF-7 cells to estrogens. An 8 h pulse with 10(-8) M E2 showed 11 polypeptides of interest, 9 displayed a transient increase in mRNA accumulation and 2 showed a temporary decreased level in the presence of this hormone. A distinct set of 2 mRNAs displayed increased amounts only after a 24 h E2 pulse.

Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase.

Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells expressing wild-type GHR (GHR1-638) or GHR truncated at amino acid 454 (GHR1-454) or 380 (GHR1-380).

Effects of estradiol-17 beta analogues on activation of estrogen response element regulated chloramphenicol acetyltransferase expression.

These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue.

Influence of estrogen structure on nuclear binding and progesterone receptor induction by the receptor complex.

The relationship between steroid structure, estrogen receptor (ER) binding affinity, nuclear binding of the ER complex, and induction of progesterone receptor (PgR) have been examined. The level of ER in membrane-free homogenates of MCF-7 cells was found to be 10.0 +/- 0.