Desensitization to gonadotropic hormones: a model system for the regulation of a G-protein-coupled receptor with 7-transmembrane spanning regions

Gonadotropic hormone, luteinizing hormone, and follicle-stimulating hormone exert their effect via activation of G-coupled receptors, which activate the hormone sensitive adenylyl cyclase, protein kinase A, and cyclic AMP responsive elements. This activation leads to specific de novo synthesis of steroidogenic factors and steroidogenic enzymes. In normal cells and following activation of this signaling pathway, desensitization period will be followed. This down-regulation, which was studied in detail for the last three decays, was found to take place at various steps of these signal transduction pathways as well as at different kinetics. A common and diverse feature of the mechanism of desensitization in other G-coupled-7-transmembrane receptor system is also discussed.     

Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle.

Crosstalk between the cyclic AMP-dependent protein kinase (PKA) and growth factor receptor signaling is one of many emerging concepts of crosstalk in signal transduction. Understanding of PKA crosstalk may have important implications for studies of crosstalk between other, less well known, signaling pathways. This review focuses on PKA crosstalk in arterial smooth muscle. Proliferation and migration of arterial smooth muscle cells (SMCs) contribute to the thickening of the blood vessel wall that occurs in many types of cardiovascular disease. PKA potently inhibits SMC proliferation by antagonizing the major mitogenic signaling pathways induced by growth factors in SMCs. PKA also inhibits growth factor-induced SMC migration. An intricate crosstalk between PKA and the mitogen-activated protein kinase (MAPK/ERK) pathway, the p70 S6 kinase pathway and cyclin-dependent kinases has been described. Further, PKA regulates expression of growth regulatory molecules. The result of PKA activation in SMCs is the potent inhibition of cell cycle traverse and SMC migration. In this review, we discuss recent advances in our understanding of the crosstalk between PKA and signaling pathways induced by growth factor receptors in SMCs, and where relevant, in other cell types in which interesting examples of PKA crosstalk have been described.     

Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739–19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)₂cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)₂cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.     

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.     

Intragonadal signalling mechanisms in the control of steroid hormone production

In the gonads, there are two recognized signal transduction mechanisms which operate in the processing of hormonal stimuli. The gonadotropins, follicle stimulating hormone and luteinizing hormone, act primarily through the generation of cyclic AMP. Several other hormonal regulators in the ovary and the testis, such as gonadotropin releasing hormone and prostaglandin F₂ stimulate inositol lipid metabolism following receptor binding. This triggers a cascading mechanism which ultimately results in the generation of increased cytosolic free calcium levels, enhanced protein kinase C activity, and liberation of arachidonic acid. There is also evidence that luteinizing hormone shares in the activation of this pathway. In this review, the significance of these signal transduction pathways is discussed in relation to the effects of various hormones on steroid biosynthesis in the gonads.     

The activation of protein kinase A by cyclic AMP is influenced by oestradiol and progesterone in supernatants from the anterior pituitary but not from hypothalamus of the female rat

Summary— The activation of protein kinase A (PKA) by cAMP was estimated in supernatant fractions from the hypothalamus (Hyp) and anterior pituitary (AP) of the female rat during the oestrous cycle and in ovariectomized and ovariectomized, ovarian steroid hormone treated animals. In both structures, the largest activation of PKA was found in dioestrus-2, while the lowest one was in Hyp in dioestrus-1 and in AP in oestrus. Ovariectomy had no influence on cAMP-dependent activation of PKA from Hyp and AP. Treatment of ovariectomized rats with 17-β-oestradiol (E₂), progesterone (P) or both abolished the activation of PKA by cAMP from AP and had no effect on hypothalamic PKA. These results indicate that ovarian steroids act specifically on AP processes via cAMP dependent pathway and regulation of PKA activity.     

Cross-talk between G protein-coupled and epidermal growth factor receptors regulates gonadotropin-mediated steroidogenesis in Leydig cells

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.     

8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein

Elevation of intracellular 8-bromo-cyclic AMP (cAMP) can activate certain steroid receptors and enhance the ligand-dependent activation of most receptors. During ligand-independent activation of the chicken progesterone receptor (cPR(A)) with the protein kinase A (PKA) activator, 8-bromo-cAMP, we found no alteration in cPR(A) phosphorylation (W. Bai, B. G. Rowan, V. E. Allgood, B. W. O'Malley, and N. L. Weigel, J. Biol. Chem. 272:10457-10463, 1997). To determine if other receptor-associated cofactors were targets of cAMP-dependent signaling pathways, we examined the phosphorylation of steroid receptor coactivator 1 (SRC-1). We detected a 1.8-fold increase in SRC-1 phosphorylation in transfected COS-1 cells incubated with 8-bromo-cAMP. Phosphorylation was increased on two mitogen-activated protein kinase (MAPK) sites, threonine 1179 and serine 1185. PKA did not phosphorylate these sites in vitro. However, blockage of PKA activity in COS-1 cells with the PKA inhibitor (PKI) prevented the 8-bromo-cAMP-mediated phosphorylation of these sites. Incubation of COS-1 cells with 8-bromo-cAMP resulted in activation of the MAPK pathway, as determined by Western blotting with antibodies to the phosphorylated (active) form of Erk-1/2, suggesting an indirect pathway to SRC-1 phosphorylation. Mutation of threonine 1179 and serine 1185 to alanine in COS-1 cells coexpressing cPR(A) and the GRE(2)E1bCAT reporter resulted in up to a 50% decrease in coactivation during both ligand-independent activation and ligand-dependent activation. This was due, in part, to loss of functional cooperation between SRC-1 and CREB binding protein for coactivation of cPR(A). This is the first demonstration of cross talk between a signaling pathway and specific phosphorylation sites in a nuclear receptor coactivator that can regulate steroid receptor activation.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A

Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways.