We investigate the receptor-mediated mechanisms by which peptide hormones activate the signaling pathways and functional responses of their target cells. We are interested in the characterization of the structure-function properties, signal transduction, and cellular processing of specific G protein–coupled receptors (GPCRs). Specifically, our research program focuses on GPCRs for the hypothalamic peptide hormone gonadotropin-releasing hormone (GnRH) and the vasoactive octapeptide angiotensin II (Ang II) and on the intracellular signaling pathways that mediate their cellular actions. The GnRH decapeptide mediates the neural control of the pituitary gland and gonadotropin secretion and is essential for normal reproductive function in both sexes. The angiotensin octapeptide plays important roles in aldosterone secretion, control of sodium balance, and regulation of blood pressure and has been increasingly implicated in the etiology of vascular and renal disease, diabetes, and cancer. We study the receptors and their functions in normal and immortalized hypothalamic neurons, pituitary gonadotrophs, adrenal glomerulosa cells, and C9 hepatic cells to elucidate their signaling pathways and the manners in which they regulate the metabolic, secretory, and growth responses of their respective target cells.
Mechanism of pulsatile neuropeptide secretion from GnRH neurons
Krsmanovic, Hu, Wada, Martinez-Fuentes,3 Navarro,4 Catt
The 1,000 or so hypothalamic GnRH neurons that control mammalian reproduction are interconnected to form a pulse generator that episodically releases GnRH at the median eminence to regulate gonadotropin secretion from the pituitary gland. Our studies on hypothalamic GnRH neurons and GT1-7 cells, the neurons’ immortalized counterpart, have established that the pulsatile secretion of GnRH is generated by an episodic autocrine interaction between GnRH and its endogenous G protein–coupled receptors expressed in GnRH-producing neurons. In addition, our studies have demonstrated that the secretion mechanism is operative in the early GnRH neurons of 13-day fetal rats before the neurons migrate from the forebrain to the hypothalamus. Beyond providing an intrinsic mechanism for regulated neurosecretion, pulsatile secretion may promote gene expression and the migration of embryonic GnRH neurons to the hypothalamus. The firing rate of GnRH neurons and the frequency of their pulsatile secretion are augmented by GnRH agonists but are diminished by rising GnRH agonist levels and abolished by GnRH antagonists and, in perifused neuronal cultures, are highly dependent on increases in intracellular Ca2+ and cyclic AMP.
The endogenous GnRH receptor is coupled to Gq, Gs, and Gi, three G proteins that mediate inositol phosphate/Ca2+/cAMP signaling. In GnRH neurons, rising GnRH concentrations cause a switch in coupling from Gq and Gs to Gi, with transient inhibition of secretory activity. Furthermore, receptor antagonists abolish pulsatile GnRH secretion. In contrast, GnRH antagonist and pertussis toxin augment Galpha(i3), with a concomitant loss of pulsatile GnRH secretion. Thus, an agonist concentration–dependent switch in the coupling of GnRHR between specific G proteins modulates neuronal signaling via Gq/Gs-stimulatory and Gi-inhibitory mechanisms. By regulating membrane ion currents, the latter may also suppress GnRH neuronal firing and episodic secretion. This autocrine mechanism serves as a timer to determine the frequency of pulsatile GnRH release by regulating Ca2+- and cAMP-dependent signaling and GnRH neuronal firing. Currently, we are studying (1) the extent to which activation of GIRK channels is involved in the mechanism of pulsatile GnRH release and (2) the role of beta/gamma subunits in regulating the process of neurosecretion at the plasma membrane.
The positive and negative feedback actions of estrogen on GnRH neurons are major regulators of GnRH secretion. Estrogen receptor (ER) alpha and beta isoforms are present in the nucleus, cytoplasm, and plasma membrane of GnRH neurons. In GT1-7 neurons, picomolar estradiol concentrations reduce cAMP production, but nanomolar concentrations augment it. The physical and functional coupling of the plasma membrane ERalpha isoform to an inhibitory G protein inhibits cAMP production. Picomolar estradiol levels increase the GnRH peak interval, shorten peak duration, and increase peak amplitude. Thus, occupancy of the plasma membrane–associated ERs expressed in GT1-7 neurons by physiological estradiol levels activates a Gi protein and modulates cAMP signaling and neuropeptide secretion.
Serotonin (5-HT) activates the GnRH neurons of the inositol 1,4,5-triphosphate/calcium signaling pathway and both stimulates and inhibits cAMP production and GnRH release. The similarity between the signaling and secretory responses elicited by GnRH and serotonin prompted us to analyze the actions of specific 5-HT receptor subtypes in deconvoluting the complex actions of these agonists on signal transduction and GnRH release. We identified specific mRNA transcripts for 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7 in immortalized GnRH neurons (GT1-7) and found that, during activation of the Gi-coupled 5-HT1A receptor, the rate of firing of spontaneous action potentials by hypothalamic GnRH neurons was profoundly inhibited as was cAMP production and pulsatile GnRH release in GT1-7 cells. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor increased the firing rate of spontaneous action potentials, stimulated InsP3 production, and caused a delayed increase in GnRH release. Selective activation of the Gs-coupled 5-HT4 receptor also increased the rate of firing of APs, stimulated cAMP production, and caused a sustained and robust increase in GnRH release. The ability of 5-HT receptor subtypes expressed in GnRH neurons to activate single or several G proteins in a time- and dose-dependent manner differentially regulates the phospholipase C (PLC)/InsP3/Ca2+ and adenylyl cyclase (AC)/cAMP signaling pathways, thereby regulating the frequency and amplitude of pulsatile GnRH release and, by modulating spontaneous electrical activity of the GnRH neuron, contributing to the control of the pulsatile neuropeptide secretion that is characteristic of GnRH neurons in vivo and in vitro.
Krsmanovic LZ, Mores N, Navarro CE, Arora KK, Catt KJ. An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc Natl Acad Sci USA 2003;100:2969-2974.
Martinez-Fuentes AJ, Hu L, Krsmanovic L, Catt KJ. Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion. Mol Endocrinol 2004;18:1808-1817.
Wada K, Hu L, Mores N, Navarro CE, Fuda H, Krsmanovic LZ, Catt KJ. 5-HT receptor subtypes mediate specific modes of serotonin-induced signaling and regulation of neurosecretion in GnRH neurons. Mol Endocrinol 2005 [Epub ahead of print].
Agonist-induced MAP kinase signaling in GnRH neurons
Shah, Catt
As in the case of many other GPCRs, activation of the GnRH-R regulates the duration and magnitude of MAP kinase phosphorylation, which in turn promotes gene expression and other intracellular responses GT1-7 neurons also express the EGF receptor (EGF-R) and respond to EGF stimulation with transient activation and phosphorylation of the EGF-R and ERK1/2. In these cells, phosphorylation and nuclear translocation of the ERK1/2-dependent protein RSK-1 accompanied phosphorylation of the EGF-R; such EGF-R phosphorylation to proceed by a pathway that is dependent on Ca2+, PKC-alpha PKC-epsilon, Pyk2, and Src. GnRH-induced transactivation of the EGF-R in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. Such transactivation of the tightly regulated EGF-R can account for the transient ERK1/2 responses that are elicited by stimulation by GnRH and certain other GPCR agonists. Agonist activation of the GnRH enhanced the association of Src with PKC-alpha and PKC-epsilon, Pyk2, and the EGF-R. As a consequence of metalloprotease activation, GnRH stimulation of GT1-7 cells causes the release/shedding of the soluble ligand HB-EGF. In particular, metalloprotease inhibition abolishes GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, RSK-1. The signaling characteristics of HB-EGF closely resemble those of GnRH and EGF with regard to the phosphorylation of the EGF-R, Shc, ERK1/2, and RSK-1 as well as with respect to the nuclear translocation of RSK-1. The findings demonstrate that GnRH-induced transactivation of the EGF-R and its downstream pathways, including ERK1/2 phosphorylation, results from ectodomain shedding of HB-EGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.
The consequences of GPCR-induced phosphorylation of MAP kinases, through transactivation of the EGF-R, include increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist–stimulated PI3K activation is not known. To clarify the issue, we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent on and/or independent of EGF-R transactivation in specific cell types. As noted above, GT1-7 cells express endogenous GnRH-R, and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of the AT1-R, lysophosphatidic acid (LPA), and the EGF-R caused phosphorylation of Akt by activating the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by the agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but dependent on PI3K activation. LPA signaling in the cells showed partial and complete dependence on EGF-R and PI3K, respectively. Our observations indicate that GPCR-induced ERK1/2 phosphorylation is dependent in some cell types or independent of PI3K in others and that the involvement of PI3K during ERK1/2 activation is not determined solely by agonist-induced transactivation of the EGF-R.
Shah BH, Catt KJ. GPCR-mediated transactivation of RTKs in the CNS: mechanisms and consequences. Trends Neurosci 2004;27:48-53.
Shah BH, Farshori MP, Catt KJ. Neuropeptide-induced transactivation of a neuronal EGF receptor is mediated by metalloprotease-dependent formation of HB-EGF. J Biol Chem 2004;279:414-420.
Shah BH, Farshori MP, Jambusaria A, Catt KJ. Roles of Src and epidermal growth factor receptor transactivation in ERK1/2 responses to GnRH receptor activation. J Biol Chem 2003;278:19118-19126.
Shah BH, Neithardt A, Chu DB, Shah FB, Catt KJ. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation MAP kinase by GPCRs. J Cell Physiol 2006;206:47-57.
Shah BH, Soh JW, Catt KJ. Dependence of GnRH-induced neuronal MAPK signaling on EGF receptor transactivation. J Biol Chem 2003;278:2866-2875.
Mechanisms and consequences of angiotensin AT1 receptor signaling
Shah, Mihalik, Hunyady, Olivares-Reyes, Catt
The Gq-coupled AT1R and its inositol phosphate/Ca2+/PKC signaling pathways mediate the diverse actions of Ang II in adrenal, renal, cardiovascular, and neural cells. In Ang II target cells, agonist stimulation of the AT1R causes phosphorylation of ERK1/2 via EGF-R transactivation-dependent and/or -independent pathways. In C9 hepatocytes, which express endogenous AT1R, Ang II–induced ERK1/2 activation is dependent on Src kinase and matrix metalloproteinases (MMPs). Agonist-mediated MMP activation in C9 cells causes shedding of HB-EGF and stimulation of ERK1/2 phosphorylation as well as marked phosphorylation of the EGF-R and its adapter molecule, Shc, and of ERK1/2 and its dependent protein, RSK1. We found that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.
In addition to their physiological roles in the cardiovascular system, GPCR agonists such as noradrenaline, ET-1, and Ang II are involved in the development of cardiac hypertrophy. Recent reports on targeted overexpression of the AT1R in cardiomyocytes suggest that Ang II can directly promote the growth of cardiomyocytes by transactivating EGF-R and subsequent activation MAPK kinases, a process that is mediated by MMP-catalyzed HB-EGF production. Blocking the generation of HB-EGF by MMP inhibitors or inhibiting EGF-R kinase activity with selective pharmacological inhibitors or antisense oligonucleotides protects against Ang II–mediated cardiac hypertrophy, suggesting a potential therapeutic strategy to prevent cardiac remodeling and hypertrophy and possibly progression to heart failure.
The Ang II–induced activation of AT1-Rs in hepatic C9 cells and consequent activation of ERK1/2 via phosphorylation and transactivation of the endogenous EGF-R occurs by a PKCdelta/Src/Pyk2-dependent pathway. Furthermore, we observed that EGF-induced activation of the EGF-R in C9 cells caused phosphorylation of the AT1-R; this was reduced by inhibition of PKC and PI3-kinase and was associated with a decrease in membrane-associated AT1-Rs and in inositol phosphate response to Ang I. We also observed that the putative PKC inhibitor Go6976 enhanced the basal and agonist-stimulated phosphorylation of ERK1/2 in C9 cells, but not by altering receptor binding and internalization, stimulating inositol phosphate production, or activating Pyk2 and Src tyrosine kinases. However, given that the PTP inhibitor sodium orthovanadate mimicked the effects of Go6976, it is possible that Go6976 enhanced agonist-induced tyrosine phosphorylation of the EGF-R by inhibiting protein tyrosine phosphatase (PTP). Moreover, selective blockade of EGF-R kinase by AG1478 abolished the ERK1/2 activation induced by Go6976. We conducted similar experiments in human embryonic kidney 293 cells, which express receptors for LPA and EGF but exhibit no significant cross-communication. Even though Go6976 caused a significant increase in EGF-induced tyrosine phosphorylation of the EGF-R and subsequent ERK1/2 activation, it had no such effects on LPA-induced responses. In Chinese hamster ovary cells, which express receptors for LPA but not for EGF, Go6976 also had no significant effect on LPA-induced ERK1/2 activation. The data indicate that Go6976 potentiates agonist-induced ERK1/2 activation by stimulating tyrosine phosphorylation of the EGF-R.
In hepatic C9 cells, Ang II–induced activation of the AT1-R stimulates ERK 1/2 phosphorylation by transactivating endogenous EGF-R through a PKC-delta/Src/Pyk2-dependent pathway, leading to phosphorylation of the EGF-R and its subsequent internalization. By contrast, we found that EGF-induced activation of the EGF-R in C9 cells caused phosphorylation of the AT1-R, which was prevented by inhibition of the intrinsic tyrosine kinase activity of the EGF-R by AG1478 and reduced by inhibition of PKC and phosphoinositide 3-kinase. EGF-induced AT1-R phosphorylation was associated with a decrease in membrane-associated AT1Rs and a reduced inositol phosphate response to Ang II. Agonist activation of endogenous AT1-Rs and EGF-Rs induced the formation of a multireceptor complex containing both the AT1-R and transactivated EGF-R. The dependence of these responses on caveolin was indicated by the finding that cholesterol depletion of C9 cells abolished Ang II–induced inositol phosphate production, activation of Akt/PKB and ERK1/2, and AT1-R internalization. Confocal microscopy demonstrated that caveolin-1 was endogenously phosphorylated and distributed on the plasma membrane in patches that undergo redistribution during Ang II stimulation. We observed agonist-induced phosphorylation and association of caveolin-1 with the AT1-R, consistent with a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. AG1478 abolished the EGF-induced AT1-R/caveolin association, suggesting that activation of the EGF-R promotes the association of caveolin and the AT1-R.
Olivares-Reyes JA, Shah BH, Hernández-Aranda J, García-Caballero AM, Farshori P, García-Sáinz JA, Catt KJ. Agonist-induced interactions between angiotensin AT1 and epidermal growth factor receptors. Mol Pharmacol 2005;68:356-364. [Perspective by Neve KA. Double feature at the signalplex. Mol Pharmacol 2005;68:275-278].
Shah BH, Baukal AJ, Shah FB, Catt KJ. Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor. Mol Endocrinol 2005;19:2535-2548.
Shah BH, Olivares-Reyes JA, Catt KJ. The protein kinase C inhibitor Go6976 potentiates agonist-induced mitogen-activated protein kinase activation through tyrosine phosphorylation of the epidermal growth factor receptor. Mol Pharmacol 2005;67:184-194.
Shah BH, Yesilkaya A, Olivares-Reyes JA, Chen H-D, Hunyady L, Catt KJ. Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor. J Biol Chem 2004;278:19118-19126.27 2004 06:47:37.
Role of angiotensin II in disease states
Catt, Hunyady
Angiotensin II activates a wide spectrum of signaling responses via the AT1R, responses that mediate Ang II’s physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II–induced AT1-R activation via Gq/11 stimulates phospholipases A2, C, and D and activates InsP3/Ca2+ signaling, PKC isoforms, and MAP kinases as well as several tyrosine kinases (Pyk2, Src, Tyk2, and FAK), scaffold proteins (GIT1, p130Cas, paxillin, and vinculin), receptor tyrosine kinases such as EGF-R, and the NFkappaB pathway. The AT1R also signals via G11/12 to activate phospholipases C and D, Rho kinase, and L-type Ca2+ channels and stimulates G protein–independent signaling pathways, such as beta-arrestin–mediated MAP kinases activation and the Jak/STAT pathway. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated rather than circulating Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species exerts potent growth-promoting, pro-inflammatory, and pro-fibrotic actions by activating cardiovascular cells, leucocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and pro-angiogenic activities. The recognition of Ang II’s deleterious actions has led to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to the hormone’s established therapeutic actions in essential hypertension.
Hunyady L, Catt KJ. Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol [Epub ahead of print].
1Left the section during FY 2005.
2Clinical Fellow, Pediatric and Reproductive Endocrinology Branch, NICHD
3Antonio Martinez-Fuentes, PhD, former Postdoctoral Fellow
4Carlos Navarro, MD, PhD, former Guest Researcher
COLLABORATORS
Tamás Balla, MD, PhD, Endocrinology and Reproduction Research Branch, NICHD, Bethesda, MD
Richard Hauger, MD, University of California San Diego, La Jolla, CA
László Hunyady, MD, PhD, DSc, Semmelweis University of Medicine, Budapest, Hungary
Nadia Mores, MD, Università Cattolica del Sacro Cuore, Rome, Italy
J. Alberto Olivares-Reyes, PhD, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico
For further information, contact cattk@mail.nih.gov.