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Peptide Hormone Receptors and Signal Transduction
- Kevin J. Catt, MD, PhD, Head, Section on Hormonal Regulation
- Lazar Krsmanovic, PhD, Staff Scientist
- Hung-Dar Chen, PhD, Adjunct Investigator
- Po Ki Leung, PhD, Visiting Fellow
- Hao Feng, MD, PhD, Visiting Fellow
The neuroendocrine control of reproductive function is expressed through the episodic secretion of gonadotropic hormones from the anterior pituitary gland in response to pulsatile stimulation by gonadotropin releasing hormone (GnRH) produced by a network of peptidergic neurons in the hypothalamus. The characteristic pulsatile secretion of GnRH from hypothalamic neurons depends on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The endogenous GnRH receptor (GnRH-R) expressed in native and immortalized GnRH neurons activates at least three G proteins, as indicated by the agonist-induced release of their specific α-subunits from the plasma membrane. Such coupling to a diverse array of G proteins provides GnRH neurons with several potential signaling pathways to transduce incoming signals. Reduction of membrane-associated Gαs at low nanomolar GnRH concentrations and of Gαq and Gαi3 at higher concentrations suggested that an agonist concentration–dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca2+ signaling via Gs-cAMP–stimulatory and Gi-cAMP–inhibitory mechanisms. This autocrine mechanism appears to function as a timer to determine the frequency of pulsatile GnRH release by regulating Ca2+– and cAMP–dependent signaling and GnRH neuronal firing.
The presence of several types of receptors in GT1-7 neuronal cell lines argues for a broad range of receptor-activated signaling and transduction pathways that may participate in the regulation of GnRH secretion into the portal circulation. Activation of protein kinase A (PKA) and protein kinase C (PKC) mediates GnRH gene transcription and GnRH secretion, suggesting that both Ca2+-inositol 1,4,5-triphosphate (InsP3) and cyclic adenosine 3′,5′-monophosphate (cAMP) pathways are involved in the transduction of plasma membrane signals in GnRH systems.
The relationship between pulsatile GnRH secretion and cAMP production in immortalized GnRH neurons
Calcium and cyclic AMP (cAMP) are important factors in the mechanism of episodic signaling in hypothalamic GnRH neurons. The observation that cAMP production in GT1-7 neurons is stimulated by both increased extracellular Ca2+ and the Ca2+ channel agonist BK-8644 and diminished by low extracellular Ca2+ and treatment with dihydropyridine analogs is consistent with activation and/or inhibition of the calcium-dependent adenylyl cyclase type I (ACI) expressed in these cells. The findings indicate that cAMP production in GnRH neuronal cells is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of ACI, and that Ca2+ influx–dependent activation of ACI acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production. In addition, cAMP–induced activation of cyclic nucleotide–gated channels (CNG) promotes Ca2+ entry and causes an increase in GnRH secretion.
Agonist activation of GnRH receptors leads to stimulation of phospholipase C, formation of IP3, mobilization of Ca2+ from intracellular stores, GnRH release, and cAMP production. To investigate the regulatory mechanisms underlying GnRH and cAMP pulsatile release, we modulated GnRH receptor activity in GnRH neurons by GnRH agonist and antagonist analogs and measured GnRH and cAMP production. The GnRH agonist [D-Ala6]Ag was used to evaluate cAMP's response to GnRH-R activation. Continuous exposure of perifused GT1-7 neurons to high (100 nM and 1 mM) GnRH receptor agonist analog [D-Ala6]Ag levels caused an increase in GnRH pulse amplitude and transiently stimulated cAMP production followed by prolonged inhibition. This is consistent with the findings that high GnRH concentrations caused coupling of GnRH-R to Gi and inhibited cAMP production. During treatment of perifused GT1-7 neurons with low GnRH receptor agonist concentrations (1 nM and 10 nM), the amplitude of GnRH pulses remained unchanged, with a concomitant increase in cAMP pulses. The findings indicate that, at low GnRH concentrations, GnRH receptors couple to adenylyl cyclase–stimulatory G proteins and cause an increase in cAMP production. Thus, GnRH exerts a bi-phasic effect on cAMP production, whereby low GnRH concentrations stimulate cAMP production and high GnRH concentrations inhibit cAMP production.
Inactivation of GnRH receptors by both peptide and non-peptide GnRH antagonist abolishes pulsatile GnRH release. The cessation of pulsatile GnRH release is dose-dependent and switches to a monotonic increase at high GnRH antagonist concentrations. Cessation of pulsatile cAMP release was also evident during GnRH antagonist treatment, and cAMP production monotonically increased with rising GnRH antagonist concentrations, suggesting that GnRH/GnRH-R–mediated inhibition is required to maintain both pulsatile GnRH and cAMP secretion. GnRH antagonist–induced inhibition of GnRH-R and an increase in cAMP production may account for cAMP–mediated increases in GnRH secretion. The data suggest that pulsatile cAMP secretion in GT1-7 neurons is driven by a GnRH/GnRH-R autoregulatory system, in which dose-dependent switching of GnRH-R coupling to adenylyl cyclase–stimulatory (Gs) and –inhibitory (Gi) G proteins mediates both pulsatile GnRH and cAMP secretion.
The lack of expression of cAMP receptors in mammalian cells as well as in GT1-7 neurons indicates that, in contrast to invertebrates, in which cAMP and its receptors provide for pulsatile cAMP release, the pulsatile cAMP is differently regulated in mammalian cells. Activation of phosphodiesterase (PDE) by PKA has been reported to play a role in establishing cAMP oscillations, which might constitute a biological clock for GnRH pulsatile release.
Pharmacologic blockade of PDE activity by IBMX, a non-selective PDE inhibitor, abolished pulsatile cAMP secretion and caused a monotonic increase in cAMP production. In contrast, GnRH secretion remained pulsatile with unchanged pulse frequency and increased pulse amplitude. Likewise, continuous treatment with 8-BR cAMP significantly increased basal GnRH release and pulse amplitude without altering GnRH pulse frequency. These findings indicate that cAMP pulsatility does not drive GnRH pulsatile GnRH secretion in hypothalamic GnRH neurons. In addition, the preserved fertility in mice lacking adenylyl cyclase 1 and 8 activity implies that cAMP is not essential in GnRH pulsatility.
Treatment of perifused GT1-7 neurons with the PKA inhibitor H-89 increased the pulse amplitude but not pulse frequency for both GnRH and cAMP. These data suggest that, under basal conditions, cAMP–inhibitory adenylyl cyclase V and VI are tonically activated by PKA and mediate inhibitory effects on both GnRH and cAMP secretion.
In summary, it is evident that pulsatile cAMP secretion in hypothalamic GnRH neurons is driven by a GnRH–autoregulatory system in which changes in GnRH pulse amplitude cause an asynchronous relation between GnRH and cAMP pulses. The pulses synchronize after occurrence of the major GnRH pulse, providing a resetting mechanism by which GnRH and cAMP pulses may synchronize. Inhibition of pulsatile cAMP release by PDE inhibitors did not affect pulsatile GnRH release, suggesting that cAMP does not participate in the regulatory mechanism that is essential for GnRH pulsatility. Thus, it is evident that, although cAMP is an active participant in GnRH secretion, it is not an essential component that drives pulsatile GnRH release, which is dependent on a GnRH–autoregulatory system in hypothalamic GnRH neurons.
Cellular distribution, G protein interaction, and functional characterization of GnRH receptors in immortalized GnRH neurons
G protein–coupled receptor (GPCR) systems have a multi-modular arrangement and provide cells with a large number of possibilities to transduce incoming signals. A typical cell appears to express several different GPCR genes, several different combinations of G protein subunits, and several isoforms of effector molecules that can be activated by each type of G protein. The differential expression of these proteins allows modulation of signals at different levels, resulting in signaling that is characteristic for a specific cell type. The current understanding is that protein-protein interactions determine the interaction between the G protein and a particular GPCR. However, several observations in various cells and tissues have indicated that different receptors coupling to the same G protein in a single cell can elicit distinct biochemical or cellular responses.
The endogenous GnRH-R expressed in native and immortalized (GT1-7) GnRH neurons activates at least three G proteins, as indicated by the agonist-induced release of their specific alpha-subunits from the plasma membrane. Such coupling to a diverse array of G proteins provides GnRH neurons with several potential signaling pathways to transduce incoming signals. The differential expression of these various proteins allows modulation of signals at many levels, resulting in messages that are optimal for specific cell functions. In our current studies, we examined localization of the components of the GnRH-R signal transduction system in GT1-7 expressing the murine receptor tagged with green fluorescent protein (GFP) at its C-terminus.
We monitored, in GT1-7 GnRH neurons, cellular distribution, receptor–G protein interaction, electrical activity, and signaling pathways of the murine GnRH-R tagged with GFP at its C-terminus and G protein alpha-subunits tagged with Renilla luciferase (Rluc). The green construct expressed in GT1-7 neurons localized to cell bodies and processes and, in fully differentiated bipolar neurons, was confined to a thin rim of cytosol at the plasma membrane, neuronal processes, and apparent synaptic junctions. In less differentiated neurons, GnRH-R–GFP was expressed throughout the cell body and processes. GnRH stimulation caused redistribution of GnRH-R–GFP, with movement close to the neurons' bipolar extensions, and increased intensity in in neuronal processes and in pre- and post-synaptic connections. BRET2 assay revealed a dose-dependent GnRH–induced interaction between GFP-tagged GnRH-R and Rluc-tagged Gαq, Gαs, and Gαi. Firing of spontaneous action potentials (APs), calcium signaling, and pulsatile GnRH secretion were comparable to those of intact GT1-7 neurons. These observations suggest that redistribution of GnRH-R may occur in areas of increased neuronal activity and indicate that specific subcellular localization, agonist-induced redistribution and coupling to multiple G proteins of the GnRH-R modulate electrical activity, second messengers, and neurosecretion in hypothalamic GnRH neurons. Binding of 125I-labeled GnRH provides additional evidence for the expression of GnRH-R in synaptic connections between GnRH neurons.
The type I mammalian GnRH receptor is an atypical GPCR in that it lacks the C-terminal cytoplasmic tail that is present in all other seven-transmembrane domain receptors. The absence of COOH-tail sequences may have resulted in a receptor that lacks the sequences needed to mediate rapid desensitization and internalization as well as strong plasma membrane expression (PME). Although the GnRH receptor is a typically membrane-bound receptor, earlier studies and recent findings have indicated expression of GnRH-R in the nucleus and cytosol.
Homo- and hetero-oligomerization and cellular signaling of the GnRH-receptor and GPR54 in immortalized GnRH neurons
The GnRH-R and G protein–coupled receptor 54 (GPR54) as well as their endogenous ligands, GnRH and kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis in mammals. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons and GT1-7 neurons revealed the expression of GnRH, GnRH-R, GPR54, and kisspeptin. Constitutive and agonist-induced bioluminescence resonant energy transfer (BRET) between GnRH-R and GPR54 tagged with Renilla luciferase (Rluc–tagged GnRH-R) or with green fluorescent protein (GFP), expressed in GT1-7 neurons, revealed homo- and hetero-oligomerization of the two receptors. Activation of endogenous GnRH-R in intact GT1-7 neurons caused a dose-dependent monotonic increase in cytosolic calcium ([Ca2+]i). In GT1-7 neurons transfected with Rluc–tagged GnRH-R, basal and maximal levels [Ca2+]i were significantly higher than in intact GT1-7 neurons. Further, we observed a transition from the dose-dependent monotonic increase of [Ca2+]i, in intact GT1-7 neurons, to a bi-phasic [Ca2+]i response in GT1-7 neurons expressing Rluc–tagged GnRH-R, where low GnRH concentrations caused inhibition of [Ca2+]i and high nanomolar and micromolar GnRH concentrations increased [Ca2+]i. In both intact and Rluc–transfected GnRH neurons, pretreatment with a GnRH antagonist abolished GnRH-induced [Ca2+]i response. Activation of endogenous GPR54 with kisspeptin-10 (kiss-10) caused monotonic dose-dependent inhibition of [Ca2+]i that was not sensitive to pertussis toxin (PTX). In GT1-7 neurons transfected with Rluc–tagged GPR54, treatment with kiss-10 caused a biphasic [Ca2+]i response. Inhibition of [Ca2+]i was observed at low nanomolar kiss-10, and a stimulatory effect of kiss-10 on [Ca2+]i was observed at high nanomolar and micromolar kiss-10 concentrations. The inhibitory actions of kiss-10 on [Ca2+]i in both intact GT1-7 neurons and GT1-7 neurons expressing Rluc–tagged GPR54 were abolished during concomitant activation of the GnRH-R with GnRH. The stimulatory effect of high kiss-10 concentrations on [Ca2+]i was potentiated during concomitant treatment with GnRH. In summary, the formation of GnRH-R and GPR54 homo- and hetero-oligomers in hypothalamic GnRH neurons and the modulation of [Ca2+]i by receptor number and agonist concentration may permit the fine tuning of hypothalamic GnRH neurons and the regulation of reproductive function.
- Frattarelli JL, Krsmanovic LZ, Catt KJ. The relationship between pulsatile GnRH secretion and cAMP production in immortalized GnRH neurons. Am J Physiol Endocrinol Metab 2011;300:E1022-30.
- Krsmanovic LZ, Hu L, Leung PK, Feng H, Catt KJ. Pulsatile GnRH secretion: roles of G protein-coupled receptors, second messengers and ion channels. Mol Cell Endocrinol 2010;314:158-163.
- Arellano-Plancarte A, Hernandez-Aranda J, Catt KJ, Olivares-Reyes JA. Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells. Biochem Pharmacol 2010;79:733-745.
- Louis SN, Chow L, Rezmann L, Krezel MA, Catt KJ, Tikellis C, Frauman AG, Louis WJ. Expression and function of ATIP/MTUS1 in human prostate cancer cell lines. Prostate 2010;70:1563-1574.
- Xing Y, Hu L, Feng H, Krsmanovic LZ, Catt KJ. Mechanisms of angiotensin II-induced ERK1/2 activation in fetal cardiomyocytes. Hormone Mol Biol Clin Invest 2010;2:277–286.
- Hao-Chia Chen, PhD, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
- Richard Hauger, MD, VA, University of California-San Diego, La Jolla, CA
- László Hunyady, MD, PhD, DSc, Semmelweis University of Medicine, Budapest, Hungary
- Simon Louis, PhD, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia
- William Louis, MD, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia
- Antonio Martínez-Fuentes, PhD, Universidad de Córdoba, Córdoba, Spain
- 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
- Márta Szaszák, PhD, Institut für Medizinisch Mikrobiologie und Hygiene, Universität Lübeck, Lübeck, Germany
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