Peptide Hormone Receptors and Signal Transduction

Kevin J. Catt, MD, PhD, Head, Section on Hormonal Regulation
Lazar Krsmanovic, PhD, Staff Scientist
Albert Baukal, Research Assistant
Hung-Dar Chen, PhD,Adjunct Investigator
Hao Feng, MD, PhD,Visiting Fellow
Lian Hu, MD, PhD, Visiting Fellow
Po Ki Leung, PhD, Visiting Fellow
Xing Yin, MD, PhD, Visiting Fellow
Samuel Quaynor, BA, Postbaccalaureate Fellow

We study the receptors and signaling mechanisms by which peptide hormones activate functional responses in their target cells. In particular, we characterize structure-function properties, signal transduction, and cellular processing of specific G protein–coupled receptors (GPCR). Our research program focuses on GPCRs for gonadotropin-releasing hormone (GnRH) and angiotensin II (Ang II) and on the intracellular signaling pathways that mediate the receptors’ cellular actions. The GnRH decapeptide mediates the neural control of the pituitary gland and gonadotropin secretion; it also exerts autocrine actions on the GnRH neurons and is essential for normal reproductive function. GnRH is a primary regulatory factor in the neuroendocrine control of reproduction and is released in an episodic manner from hypothalamic GnRH neurons, which, as we previously found, express GnRH receptors. The angiotensin octapeptide exerts regulatory actions on aldosterone secretion, sodium balance, and blood pressure and has been implicated in the etiology of cardiac, vascular, and renal disease as well as in diabetes mellitus. We study the regulation and functions of these receptors in normal and immortalized hypothalamic neurons, pituitary gonadotropes, adrenal glomerulosa cells, hepatic cells, and transfected cell lines in order to elucidate their signaling pathways and the mechanisms by which they regulate metabolic, secretory, and growth responses.

Expression and regulatory functions of estrogen receptor-α and -β subtypes in hypothalamic GnRH neurons

Hu, Gustofson,¹ Feng, Leung, Krsmanovic, Catt; in collaboration with Mores

Estradiol (E₂) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, exerting both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors expressed in GnRH neurons has been controversial. In our study, single-cell RT-PCR revealed the expression of both estrogen receptor alpha (ERα) and beta (ERβ) isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERα and ERβ or individual ERs were expressed in 94 percent of cultured fetal GnRH neurons. In adult female rats at diestrous, 68 percent of GnRH neurons expressed ERs, followed by 54 percent in estrous and 19 percent in proestrous. In addition, we found expression of individual ERs in 24 percent of adult male GnRH neurons. ERα exerts marked G_i-mediated inhibitory effects on spontaneous AP firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E₂ concentrations and ERβ agonists raised the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERβ-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERα to pertussis toxin (PTX)–sensitive G_i/o proteins, E₂ also activated G protein–activated inwardly rectifying potassium (GIRK) channels, thereby decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. Our findings demonstrate that the dual actions of E₂ on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERα and ERβ expressed in hypothalamic GnRH neurons.

Role of tyrosine³²⁴ of the GnRH receptor in ligand binding and cAMP signaling

Krsmanovic, Hu, Feng, Leung, Wada,² Catt

The Asn-Pro-any amino acid-Tyr (NPX_2-3Y) sequence of the seven-transmembrane (TM) GPCRs, in which X represents an aliphatic amino acid, mediates interactions with intracellular effector molecules. In the GnRH receptor, the sequence is modified to DPXXY by exchange of the Asp and Asn residues normally present in TM II and TM VII, respectively, but retains the Tyr residue that is conserved in the NPXZY sequence motif of most GPCRs. In the mouse GnRH-R, Y³²⁴ is outside the DPLIY motif and is one of only two tyrosine residues that face the side of the receptor. We mutated Tyr³²⁴ to phenylalanine to preserve the original aromatic component of the tyrosine residue. We analyzed binding properties and cAMP signaling in cells expressing endogenous GnRH-R and in COS -7 cells transfected with mouse GnRH-R. In cultured hypothalamic cells and immortalized GnRH neurons (GT1-7 cells), activation of the endogenous GnRH-R caused a biphasic cAMP response for which inhibition was G_i-dependent. Agonist stimulation of GnRH-R expressed in COS-7 cells caused a monotonic dose-dependent increase in cAMP production. In contrast to the case of neuronal cells, the stimulatory action of GnRH on cAMP in HEK-293 cells expressing the GnRH-R was PTX-sensitive, suggesting cell specificity in G_i/o activation. In COS-7 cells expressing the Tyr³²⁴F mutant receptor, maximal binding of a ¹²⁵I-labeled GnRH agonist analogue was reduced by only 10 percent. We noted that cAMP production remained monotonic and dose-dependent and that the ED₅₀ of the Tyr³²⁴F mutant receptor declined about 200 fold. In contrast to the wild-type (WT) GnRH-R, PTX pretreatment of COS-7 cells expressing the Tyr³²⁴F mutant receptor had no effect on cAMP production. Substitution of the aromatic-hydrophobic Tyr²³⁴ of GnRH-R with neutral, hydrophilic serine caused a 70 percent decrease in ¹²⁵I-labeled binding and loss of cAMP signaling. In summary, our data indicate that Tyr²³⁴ plays an important role in ligand binding, cAMP signaling, and GnRH-R–G_i/o interaction.

BRET analysis of constitutive and ligand-induced homo- and hetero-oligomerization of GnRH and GPR54 receptors

Krsmanovic, Hu, Feng, Leung, Quaynor, Wada,² Catt

Kisspeptins, a family of peptides encoded by the KiSS-1 gene, promote GnRH secretion and are endogenous ligands for the GPCR GPR54. Both KiSS-1 and GPR54 are expressed in the mammalian hypothalamus, consistent with observations that kisspeptins and GPR54 are involved in activation and regulation of the hypothalamic-pituitary-gonadal axis. Disrupted GPR54 signaling results in hypogonadotrophic hypogonadism in rodents and humans. Central or peripheral administration of kisspeptin potently stimulates the hypothalamic-pituitary-gonadal axis and increases circulating gonadotrophin concentrations in animal models. GPR54 is widely expressed in many tissues related to reproductive function, and evidence suggests that kisspeptin acts predominantly within the central nervous system to regulate GnRH secretion.

The GnRH receptor (GnRH-R) and GPR54 and their specific ligands, GnRH and kisspeptin, are essential regulators of the hypothalamic-pituitary-gonadal axis. Bioluminescence resonance energy transfer (BRET) analysis revealed constitutive and GnRH-activated BRET² signals in HEK-293 cells transfected with DNAs for GnRH-R-Rluc and GnRH-R-GFP². BRET² signaling reached a maximum at a 1:12 GFP²/Rluc ratio and was significantly increased by the GnRH-R agonist analogue [d-Ala⁶]Ag. In contrast, treatment of HEK-293 cells expressing GnRH-R homo-oligomers with kisspeptin-10, a GPR54 agonist, had no significant effect on the BRET² signal. We also observed formation of GPR54 homo-oligomers in cells transiently transfected with GPR54-Rluc cDNA and increasing amounts of GPR54-GFP² cDNA. Similarly, treatment of HEK-293 cells expressing GPR54 homo-oligomers with [d-Ala⁶]Ag reduced the BRET² signal, suggesting that direct activation of GPR54 by kisspeptin-10 or co-activation of GPR54 by [d-Ala⁶]Ag causes inhibition of homo-oligomer formation. Transfection of HEK-293 cells with GPR54-Rluc cDNA and increasing amounts of GnRHR-GFP² led to formation of GPR54/GnRH-R hetero-oligomers. Treatment of such complexes with kisspeptin-10 or GnRH-R activation by [d-Ala⁶]Ag significantly increased the BRET² signal. Constitutive and GnRH-activated increases in BRET signals occurred in HEK-293 cells expressing GnRH-R homo-oligomers. Previous treatment with GnRH antagonist prevented the GnRH-induced increase in the BRET signal. We observed GPR54 homo-oligomers when we transfected HEK-293 cells with a constant amount of GPR54-Rluc cDNA and increasing amounts of GPR54-GFP² cDNA. BRET² signal intensity decreased significantly during treatment with kisspeptin-10. Transfection of HEK-293 cells with GPR54-Rluc and increasing amounts of GnRHR-GFP² caused formation of GPR54/GnRH-R hetero-oligomers. Treatment of such complexes with kisspeptin-10, a GPR54 agonist, or selective GnRH-R activation by GnRH significantly increased the BRET² signal. Our results suggest that constitutive and agonist-mediated formation of GnRH-R and GPR54 homo- and hetero-oligomers could play a significant role in the regulation of receptor activation in hypothalamic GnRH neurons.

Hu L, Gustofson RL, Feng H, Leung PK, Mores N, Krsmanovic LZ, Catt KJ. Converse regulatory functions of estrogen receptor-α and -β subtypes expressed in hypothalamic GnRH neurons. Mol Endocrinol 2008;22:2250-2259.
Hu L, Wada K, Mores N, Krsmanovic LZ, Catt KJ. Essential role of GIRK channels in gonadotropin-induced regulation of GnRH neuronal firing and pulsatile neurosecretion. J Biol Chem 2006;281:25231-25240.
Neithardt A, Farshori MP, Shah FB, Catt KJ, Shah BH. Dependence of GnRH-induced phosphorylation of CREB and BAD on EGF receptor transactivation in GT1-7 neuronal cells. J Cell Physiol 2006;208:586-593.
Quaynor S, Hu L, Leung PK, Feng H, Mores N, Krsmanovic LZ, Catt KJ. Expression of a functional GPR54-kisspeptin autoregulatory system in hypothalamic GnRH Neurons. Mol Endocrinol 2007;21:3062-3070.
Wada K, Hu L, Mores N, Navarro CE, Fuda H, Krsmanovic LZ, Catt KJ. Serotonin (5-HT) receptor subtypes mediate specific modes of 5-HT-induced signaling and regulation of neurosecretion in gonadotropinreleasing hormone neurons. Mol Endocrinol 2006;20:125-135.

Functional interactions between the AT₁ receptor and the Ii protein

Chen H-D, Baukal, Catt; in collaboration with Chen H-C, Szaszák, Hunyady

Site-directed mutagenesis studies have defined the membrane-proximal region of the cytoplasmic tail of the AT₁ receptor (AT₁R) as a site required for normal AT₁R folding and surface expression. Using yeast two-hybrid screening of a human embryonic kidney cDNA library with the AT₁R carboxyl-terminal tail as bait, we identified the Invariant chain (Ii protein) or CD74 as a novel interacting protein. We confirmed such association with co-immunoprecipitation and co-localization studies. We localized the binding site for Ii on the AT₁R carboxyl-terminal tail to a region that is important for exit of AT₁R from the endoplasmic reticulum (ER) and that is conserved in many G protein–coupled receptors. Transient co-expression of Ii with AT₁-R in CHO (Chinese hamster ovary) cells consistently reduced AT₁R density at the cell surface. Furthermore, the interaction of Ii with the carboxy-terminal tail of AT₁R increased the receptor’s retention in the ER and promoted its proteasomal degradation. These observations indicate that Ii and AT₁R become associated in the early biosynthetic pathway and demonstrate that the Ii protein is a negative regulator of AT₁R expression.

Szaszák M, Chen HD, Chen HC, Baukal A, Hunyady L, Catt KJ. Identification of the invariant chain (CD74) as an angiotensin AGTR1 receptor-interacting protein. J Endocrinol 2008;199:165-176.

Role of angiotensin II in prostate cancer

Catt; in collaboration with Louis S, Louis W

Angiotensin II (Ang II) promotes cell growth and proliferation and has been implicated in several forms of tumorigenesis. It is present in the basal cell layer of the normal prostate gland and in benign prostatic hyperplasia (BPH) and stimulates cell growth via AT₁ receptors. Furthermore, AT₁ receptor blockers have been shown to reduce prostate-specific antigen and to inhibit prostate cancer cell growth. An analysis of Ang II expression in BPH and prostate cancer, including high-grade prostatic intraepithelial neoplasia (HGPIN), showed Ang II’s presence not only in basal epithelial cells in BPH but also in proliferating malignant cells in prostate cancer (Gleason grades 2–5) and in the cytoplasm of LNC aP, DU 145, and PC 3 prostate cancer cell lines. The data demonstrate the presence of Ang II staining in malignant cells in all grades of prostate cancer and indicate that Ang II expression in non-basal epithelial cells is an early indication of pre-malignant and malignant changes. In view of its known mitogenic activity, Ang II likely contributes to the growth and infiltration of malignant epithelial cells in the prostate. Furthermore, based on the recent observation by Baker et al. (Am J Physiol Cell Physiol 2006;291:C995) that elevated levels of cytoplasmic Ang II can increase cell proliferation via a non–AT₁R mechanism, it is possible that angiotensin-converting enzyme (ACE ) inhibitors might also be of value in the treatment of prostate cancer by reducing intracellular Ang II formation.

Louis SN, Wang L, Chow L, Rezmann LA, MacGregor DP, Casely D, Catt KJ, Frauman AG, Louis WJ. Appearance of angiotensin II expression in non-basal epithelial cells is an early feature of malignant change in the human prostate. Cancer Detect Prev 2008;31:391-395.

Role of caveolin in receptor signaling

Yin, Chen H-D, Catt

Caveolin1 (Cav1) is an important component of the plasma-membrane microdomains, such as caveolae and lipid rafts, that are associated with AT₁ and epidermal growth factor (EGF) receptors in certain cell types. An analysis of the interactions between Cav1 and other signaling molecules that mediate AT₁R function in Ang II– and EGF-stimulated hepatic C9 cells demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular Ca²⁺ in cells stimulated by Ang II, but not by EGF, and that Cav1 plays a scaffolding role in the process of MAPK (mitogen-activated protein kinase) activation. Furthermore, intracellular Ca²⁺ and Src regulate Cav1 phosphorylation by Ang II and epidermal growth factor (EGF). Phosphorylation of Cav1 and epidermal growth factor receptor (EGFR) by Ang II, but not extracellular signal-regulated kinase (ERK)1/2 activation, is dependent on intracellular Ca²⁺. The PI 3-kinase inhibitors LY294002 and wortmannin differentially modulate both Cav1 and EGFR activation by Ang II through intracellular Ca²⁺. Our findings further demonstrate the importance of Cav1 in conjunction with receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways operate in Ang II– and EGF-stimulated C9 cells and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.

Hunyady L, Catt KJ. Pleiotropic AT₁ receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol 2006;20:953-970.
Olivares-Reyes JA, Shah BH, Hernández-Aranda J, García-Caballero A, Farshori MP, García-Sáinz JA, Catt KJ. Agonist-induced interactions between angiotensin AT₁ and epidermal growth factor receptors. Mol Pharmacol 2005;68:356-364.
Yin X, Li B, Chen H, Catt KJ. Differential signaling pathways in angiotensin II- and epidermal growth factor signaling in LNcaP and PC3 cancer cells. Mol Pharmacol 2008;74:1223-1233.

Role of Ang II in insulin resistance

Catt; in collaboration with the Olivares-Reyes research group

Insulin resistance is an important factor in the development of Type 2 diabetes mellitus (DM2). At the molecular level, insulin resistance is linked to reduced tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1). The mechanisms of this process have not been completely identified, but the role of serine/threonine phosphorylation of IRS-1 in desensitization of insulin action is well established. Studies performed in collaboration with Alberto Olivares-Reyes revealed that blockade of the renin-angiotensin system by inhibiting either ACE or the Ang II AT₁ receptor reduces insulin resistance and may decrease DM2 risk. We examined the interactions between the Ang II and insulin signaling systems in hepatocytes as well as the regulation of IRS-1 phosphorylation and insulin-Akt activation by Ang II in clone 9 (C9) hepatocytes. In C9 cells, Ang II specifically desensitized insulin-induced Akt Thr308 activation. The inhibition was associated with increased IRS-1 phosphorylation on Ser636/Ser639 that was prevented by inhibition of EGFR tyrosine kinase activity. Insulin-induced phosphorylation of IRS-1 on Ser636/639 is mediated mainly by PI3K/mTOR/S6K-1. Inhibition of PI3K (by wortmannin) and mTor (by rapamycin) revealed that Ang II stimulates IRS-1 phosphorylation on Ser636/639 via the PI3K/mTOR/S6K-1 pathway. Both agents also blocked the inhibitory effect of Ang II on insulin-induced activation of Akt. Our studies suggested that MAPK/S6K-1 is a secondary pathway involved in the Ang II–induced inactivation of insulin signaling. In summary, Ang II inhibits insulin signaling by increasing ser636/ser639 IRS phosphorylation via a mechanism dependent on EGFR transactivation that leads to MAPK/S6K1 and PI3-K/Akt/mTor/S6K-1 activation. Our findings indicate that Ang II plays a significant role in the development of insulin resistance via the EGFR.

Kisspeptin/GPR54 in the endocrine pancreas

Catt; in collaboration with the Martínez-Fuentes research group

As discussed above, the KiSS-1/GPR54 system plays a major role in the regulation of the gonadotropic axis by stimulating GnRH secretion at the hypothalamic level and activating gonadotropin release. The KiSS-1/GPR54 system is expressed in both human and mouse pancreas, indicating that the neuroendocrine actions of KiSS-1 and GPR54 are not restricted to the hypothalamic/pituitary system and might participate in the regulation of pancreatic islet function. Studies conducted with Antonio Martínez-Fuentes and colleagues confirmed the expression of KiSS-1 and GPR54 transcripts in the rat pancreas and Ins-1 beta cells. Analysis of the direct effects of kisspeptin-10 on calcium kinetics in cultured beta cells in a microfluorimetric system revealed that 70 percent of insulin-producing cells responded with a prominent calcium increase. Given the coupling between calcium increase and exocytosis, the data suggested that kisspeptin directly induces insulin release in pancreatic beta cells. Furthermore, kisspeptin-10 administration caused a significant increase in FM5-95 incorporation in single beta cells, similar to that elicited by glucose administration, thus supporting a role for kisspeptins in insulin secretion. Analysis of KiSS-1 and GPR54 expression under high glucose conditions revealed a significant reduction in the number of KiSS-1 transcripts while transcripts for GPR54 remained unaltered. In summary, the presence of KiSS-1 and GPR54 transcripts suggests that endogenous kisspeptins may regulate insulin production through an autocrine mechanism in pancreatic beta cells. Furthermore, regulation of their expression by one of the main stimuli of insulin secretion supports the participation of the KiSS-1/GPR54 system in the pathophysiology of the endocrine pancreas.

¹ Robert Gustofson, MD, former Clinical Fellow
² Keiko Wada, MD, PhD, former Postdoctoral Fellow

Collaborators

Hoa-Chia Chen, PhD, Program in 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, Institute of Medical Biology and Hygiene, Universität Lübeck, Lübeck, Germany

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