You are here: Home > Section on Hormonal Regulation

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
Anli Tong, MD, PhD, Volunteer

This program investigates the mechanisms of action of regulatory peptide hormones in specific endocrine systems, mainly those that mediate reproductive hormone secretion (GnRH) and cardiovascular function (Angiotensin II). The research includes analysis of two specific G protein–coupled receptors (GPCRs) that bind to GnRH and the angiotensin II octapeptide.

The mechanism of GnRH pulse generation, which is an intrinsic property of the GnRH neuron, is a major subject of our research program. Studies on GnRH neuronal membrane excitability focus on hyperpolarization-activated inward current (Ih). Inward rectifier potassium channels (Kir) modulate cell excitability by determining the resting potential, potassium homeostasis, and repolarization of the cell membrane. These studies are highly relevant to our project and the understanding of pulsatile GnRH release. Our research also addresses the homo- and hetero-oligomerization of GPCRs expressed in GnRH neurons. Ligand-binding studies and measurements of second messengers were used to provide insights into the physiological functions of GPCR homodimers and heterodimers. We also monitored changes in the affinity of the receptors for specific ligands, as well as the mechanism by which one receptor in a heterodimer can alter the binding of a ligand specific for the pairing receptor. We used bioluminescence resonance energy transfer (BRET) to monitor GnRH-R– and GPR54-induced activation of specific G proteins, and to detect basal precoupling and agonist-promoted interactions of these receptors with Gα-, Gβ- and Gγ-subunits of G_q, G_s, and G_i/o G proteins. These approaches have provided further insights into the interactions between receptor and Gαβγ-complexes. Protein phosphorylation has emerged as one of the fundamental mechanisms of signal transduction in many cells. Activation of the PLC family, including β, γ, δ, and ϵ, produces two second messengers: inositol 1,4,5-P₃, (InsP₃) and diacylglycerol (DAG), which elicit cellular responses through a variety of effectors. Protein kinase D (PKD), a member of a novel family of serine/threonine protein kinases, has a unique position in the signal transduction pathways initiated by DAG and protein kinase C (PKC). PKD is not only a direct DAG target but also acts downstream of PKCs in a novel signal transduction pathway that is implicated in the regulation of multiple fundamental biological processes such as Golgi structure and function, sorting of membrane proteins, and exocytosis.

GnRH-induced modulation of electrical activity in GnRH neurons

The majority of recorded GnRH neurons showed irregular AP firing, with transition between slow, high spike amplitude tonic firing, and intervals of fast, lower spike-amplitude, burst-like AP firing. Transition from slow to fast tonic AP firing was associated with the appearance of switching APs that are characterized by fAHP, threshold afterdepolarization, and initiation of fast AP firing. Treatment of hypothalamic GnRH neurons with nanomolar GnRH concentrations increased the occurrence of fast tonic APs, and reduced the duration of mAHP current. In contrast, treatment of hypothalamic GnRH neurons with micromolar GnRH concentrations abolished the appearance of fast tonic AP firing and did not affect the occurrence of slow tonic AP firing. The inhibitory action of high GnRH concentrations on AP firing was prevented by PTX and the underling currents were identified as GIRK currents. These responses indicate that agonist-stimulation of endogenous GnRH receptors expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. The data indicate that AP- and GnRH-driven Ca²⁺ influx, and coupling of GnRH-R to G_i/o in GnRH neurons, determine the profile of after-hyperpolarization currents and consequently mediate firing frequency and the spike-profile. GnRH-induced modulation of Ca²⁺ influx and the consequent changes in AHP current suggest that the GnRH receptors expressed in hypothalamic GnRH neurons are important modulators of their neuronal excitability. Endogenous GnRH receptors expressed in native and immortalized GnRH neurons initiate diverse signaling pathways by coupling to multiple G proteins. Such coupling is time- and dose-dependent, and can switch between G_q, G_s, and G_i/o, in GnRH-stimulated GT1-7 neurons. The findings suggest that an agonist concentration–dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca²⁺ signaling via G_s-cAMP–stimulatory and G_i-cAMP–inhibitory mechanisms. Activation of Gimay also inhibit GnRH neuronal function and episodic secretion by regulating membrane ion currents, possibly through activation of G protein–regulated inwardly rectifying potassium channels (GIRKs). Such channels are important for maintaining the resting potential and excitability of neurons. Accordingly, it is necessary to clarify the functional activities of these channels, activities that are associated with regulation of the GnRH neuron, as well as the neuron's spontaneous electrical activity and episodic neuropeptide secretion.

Action potentials and the associated Ca²⁺ influx are followed by slow afterhyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca²⁺-dependent K⁺ current. Slow AHPs are common in mammalian neurons and are present in both peripheral and central nervous systems. The firing of individual, and/or bursts, of APs in spontaneously active GnRH neurons is followed by hyperpolarization that lasts from several milliseconds (ms) to several seconds (s). Such hyperpolarization is mediated by the activation of two families of Ca²⁺-activated K⁺ channels. Big conductance (BK) channels contribute to AP repolarization, whereas small conductance (SK) channels underlie the AHP and mediate firing frequency and spike-frequency adaptation. Neither fast AHPs (fAHP) nor sAHPs were observed during spontaneous firing of fast rhythmic APs. Small calcium-activated potassium current was recorded as a tail current by a depolarization step from −60 mV to 40 mV. Agonist activation of GnRH neurons caused a significant increase in sAHP that was partially sensitive to apamin. Also, treatment of GnRH neurons with 10 nM GnRH increased the occurrence of high-frequency broad APs, with unchanged decay constants for fAHP and mAHP. In contrast, treatment of GnRH neurons with 1 µM GnRH abolished mAHP current, but did not affect the occurrence of fAHP current. This was followed by subthreshold after-depolarization potential (ADP) and significant reduction of the AP firing frequency. The data indicate that AP- and GnRH-driven Ca²⁺ influx in GnRH neurons determines the profile of AHP currents and consequently mediates firing frequency and the spike-profile.

Expression of a GPR54-kisspeptin autoregulatory system in GnRH neurons

The G protein–coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons revealed expression of GnRH, kisspeptin-1, and GPR54. Also, constitutive and GnRH agonist–induced BRET between the Renilla luciferase–tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in HEK 293 cells, revealed hetero-oligomerization of the two receptors. Similar to other G protein–coupled receptors in which hetero-oligomer formation influences binding and/or signaling properties, the formation of GnRH-R-GPR54 hetero-oligomers may provide for integrated cellular signaling that regulates GnRH and kisspeptin secretion from GnRH neurons. The inhibition of kisspeptin secretion by GnRH suggests that its activation of the heterodimeric GnRH-R/GPR54 receptor complex favors selective coupling to the G_i/o signaling pathway. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion suggest that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control GnRH and kisspeptin secretion from GnRH neurons. The distribution of GnRH-R-GFP is comparable to that seen in GT1-7 neurons expressing the native GnRH-R; GnRH-R-GFP is localized to cell bodies and processes, and, in fully differentiated bipolar neurons, is confined to a thin rim of cytosol at the plasma membrane and in neuronal processes. GnRH stimulation causes redistribution of GnRH-R-GFP, with movement in close proximity to their bipolar processes and in the area of apparent synaptic connections. GnRH-Rs expressed in native and GT1-7 neurons form homo-oligomers and activate diverse signaling pathways by coupling to at least three G proteins. Such coupling is time- and dose-dependent and switches between G_q, Gs, and G_i.

GnRH-mediated activation of protein kinase D in immortalized GnRH neurons

GnRH receptors (GnRH-Rs) expressed in native and immortalized GnRH neurons (GT1-7) and pituitary gonadotrophs (αT3) activate diverse signaling pathways by coupling to at least three G proteins. Such coupling is time- and dose-dependent, and switches between G_q, G_s, and G_i/o according to the agonist concentration. These findings suggest that an agonist concentration–dependent switch in coupling of the GnRH-R between specific G proteins modulates diverse signaling pathways in these cells types. Protein phosphorylation has emerged as one of the fundamental mechanisms of signal transduction in many cells. Activation of the PLC family, including β, γ, δ, and ϵ, produces two second messengers: inositol 1,4,5-P₃, (InsP₃) and diacylglycerol (DAG), which elicit cellular responses through a variety of effectors. Protein kinase D (PKD), a member of a novel family of serine/threonine protein kinases, has a unique position in the signal transduction pathways initiated by DAG and protein kinase C (PKC). PKD is not only a direct DAG target but is also downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes such as Golgi structure and function, sorting of membrane proteins, and exocytosis. GnRH-induced activation of GnRH-R in both GT1-7 neurons and αT3 gonadotrophs caused stimulation of PKD. GnRH-induced PKD phosphorylates both Ser742/Ser744 and Ser 916 in a time- and dose-dependent manner. This activation is abolished by a GnRH-R antagonist, consistent with GnRH-R-mediated activation. The PKC inhibitor Gö 6983 abolishes phorbol 12-myristate13-acetate–induced PKD phosphorylation, but only partly inhibits GnRH-induced PKD Ser742/744 activation. In contrast, GnRH-induced PKD activation was unaffected by the PKC-specific antagonist Gö 6976. These findings indicate that, in addition to PKC, the agonist-stimulated GnRH-R also activates PKD, providing a mechanism of signal integration and amplification. In conclusion, the data suggest that GnRH-GnRH-R–induced activation of PKD in immortalized GnRH neurons and pituitary gonadotrophs causes complex molecular interactions that maintain GnRH and LH secretion from the hypothalamo-pituitary axis.

Role of renin-angiotensin system in prostate cancer

The renin-angiotensin system (RAS) has important regulatory actions on growth factors and cell growth. There is emerging evidence that the incidence of cancer is reduced in patients undergoing long-term treatment with drugs that inhibit the RAS, with reports suggesting that Ang II directly stimulates cell growth via the AT₁-receptor and that ACE inhibition or AT₁-receptor blockade inhibits the growth of a range of tumors, including prostate cancer. All components of the RAS have been identified in the prostate including Ang II, which has been localized to the basal epithelial cells in normal human prostate and to malignant epithelial cells in prostate cancer biopsies. More recently, we demonstrated the existence of functional AT₂-receptors, which inhibit the proliferative effects of EGF on DNA synthesis and MAPK phosphorylation, in both early stages, androgen-dependent, LNCaP, and late stage, androgen-independent PC3 prostate cancer cell lines. Therefore, it is possible that the reported anti-proliferative action of AT₁-receptor blockers in prostate cancer is not due solely to the blockade of AT₁-receptor stimulation, but may also be in part attributable to endogenous Ang II selectively activating the AT₂-receptor in the presence of AT₁-blockade.

Modulation of insulin signaling by angiotensin II

One of the major targets of the activated Akt kinase is GSK-3, which has an important role in the regulation of glycogen synthesis via inhibitory phosphorylation of glycogen synthase (GS). Upon insulin-mediated phosphorylation on Ser^21/9 of the two isoforms of GSK-3—GSK-3α and GSK-3β—GSK-3 is inactivated. This inactivation, in parallel with protein phosphatase-1 activation, relieves the inhibitory phosphorylation of GS, which becomes activated and promotes glycogen synthesis. To evaluate the metabolic impairment caused by Ang II as a consequence of its inhibitory effect on Akt, we determined its effect on insulin-induced GSK-3α/β Ser^21/9 phosphorylation. Our findings indicate that Ang II impairs insulin-induced Akt Thr³⁰⁸ phosphorylation by increasing IRS-1 Ser⁶³⁶/Ser⁶³⁹ phosphorylation and IRS-1 protein degradation, by a mechanism dependent on EGF transactivation that leads to PI3K/ERK1/2/mTOR/S6K-1 activation, providing evidence that defines a role of Ang II in the development of insulin resistance.

Angiotensin II-induced ERK1/2 activation in fetal cardiomyocytes

Growth factors such as Ang II are known to stimulate cAMP-dependent products of PKA via G_s-adenylate cyclase interactions. PKA might regulate ERK1/2 through Raf-dependent or -independent signaling pathways, according to cell type. Stimulation with Ang II increases intracellular cAMP production and PKA activation solely via the AT₁ receptor; in turn, PKA can negatively regulate Ang II–induced ERK1/2 activation via unknown intermediate signaling molecules, but not c-Raf and/or the EGFR, in fetal cardiomyocytes. In most cell types, including cardiomyocytes, PLC has been reported to mediate the production of inositol trisphosphate (IP₃) and diacylglycerol, which results in PKC activation during Ang II receptor signaling. In particular, PLCβ1, as one of PLCβ1-4, appears to be mainly expressed in the heart and to have unspecified roles in cellular proliferation and differentiation. In AT₂ receptor signaling, ERK1/2 is phosphorylated through c-Raf, which is positively activated by unknown intermediate signaling molecules, but not via EGFR transactivation. In AT₁ receptor signaling, EGFR transactivation is required for ERK1/2 phosphorylation through c-Raf, which is consistent with our previous report that defined the roles of EGFR in Ang II–induced ERK1/2 activation in other cell types. The present studies reveal that multiple and parallel signaling pathways are involved in the mechanism of signaling pathways of Ang II–induced ERK1/2 activation in fetal cardiomyocytes. The results are likely to account for balancing the contributions of such multiple signaling pathways to Ang II–induced ERK1/2 activation in fetal cardiomyocytes.

Publications

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 1-70(14):1563-1574
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
Chow L, Rezmann L, Catt KJ, Louis WJ, Frauman AG, Nahmias C, Louis SNS. Role of the renin-angiotensin system in prostate cancer. Mol Cell Endocrinol 2009 302:219-229
Xing Y, Hu L, Feng H, Krsmanovic LZ, Catt KJ. Mechanisms of Angiotensin II-Induced ERK1/2 Activation in Fetal Cardiomyocytes. Hormone Molecular Biology and Clinical Investigation 2010 [E-pub ahead of print]
Krsmanovic LZ, Hu L, Leung P-K, Feng H, Catt KJ. The hypothalamic GnRH pulse generator: multiple regulatory mechanisms. Trends Endocrinol Metab 2009 8:402-408

Collaborators

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

Contact

For further information, contact cattk@mail.nih.gov.

Top of Page