PEPTIDE HORMONE RECEPTORS AND SIGNAL TRANSDUCTION
, PhD, Head, Section on Hormonal Regulation
Lazar Krsmanovic, PhD, Staff Scientist
Albert Baukal, Research Assistant
Hung-Dar Chen, PhD, Adjunct Investigator
Hao Chia Chen, PhD, Facility Manager
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 specific target cells. This work includes the characterization of structure-function properties, signal transduction, and cellular processing of specific G protein–coupled receptors (GPCRs). Our research focuses on GPCRs for gonadotropin-releasing hormone (GnRH) and angiotensin II (Ang II) and the intracellular signaling pathways that mediate their cellular actions. The GnRH decapeptide mediates neural control of the pituitary gland and gonadotropin secretion, 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 we previously found to express GnRH receptors. The angiotensin octapeptide exerts regulatory actions on aldosterone secretion, control of sodium balance, and regulation of blood pressure and has been implicated in the etiology of cardiac, vascular, and renal disease as well as in diabetes mellitus. To elucidate the signaling pathways of these receptors and the mechanisms by which they regulate metabolic, secretory, and growth responses, we study their regulation and function in normal and immortalized hypothalamic neurons, pituitary gonadotropes, adrenal glomerulosa cells, hepatic cells, and transfected cell lines
Regulatory mechanisms controlling pulsatile GnRH secretion
Our previous studies in native and immortalized (GT1-7) hypothalamic GnRH neurons showed that the autocrine actions of GnRH on its cells of origin elicit both inhibitory and stimulatory responses as a consequence of the activation of several G proteins; these actions are essential for episodic GnRH secretion. In addition, we found that GnRH neurons express G protein–gated, inwardly rectifying potassium (GIRK) channels that are activated by luteinzing hormone/human chorionic gonadotropin (LH/hCG), leading to inhibition of membrane excitability and pulsatile GnRH secretion (Hu et al., J Biol Chem 2006;281:25231). More recently, we found that the GPR54 receptor and its endogenous ligand, kisspeptin, which are both essential for activation and regulation of the hypothalamic-pituitary-gonadal axis, are expressed in hypothalamic GnRH neurons and GT1-7 cells. Furthermore, constitutive and GnRH agonist–induced bioluminescence resonance energy transfer [BRET(2)] between Renilla luciferase (Rluc)–tagged GnRH-R and GPR54 labeled with green fluorescent protein [GFP(2)], expressed in HEK-293 cells, revealed hetero-oligomerization of the two receptors. The initial depolarizing effects of kisspeptin on membrane potential caused increased neuronal firing and increased GnRH peak amplitude and duration. Treatment with GnRH significantly reduced kisspeptin production and secretion in hypothalamic GnRH neurons and GT1-7 cells. These findings suggest that kisspeptins can act as paracrine and/or autocrine regulators of the GnRH neuron. Both the stimulation of GnRH release by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control the production and secretion of both GnRH and kisspeptin from GnRH neurons.
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;208:586-93.
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;21:3062-70.
Shah BH, Neithardt A, Chu DB, Shah FB, Catt KJ. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol 2006;206:47-57.
Shah BH, Shah FB, Catt KJ. Role of metalloproteinase-dependent EGF receptor activation in alpha-adrenoceptor-stimulated MAP kinase phosphorylation in GT1-7 neurons. J Neurochem 2006;96:520-32.
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 gonadotropin-releasing hormone neurons. Mol Endocrinol 2006;20:125-35.
Functional interactions between the angiotensin AT1 receptor and the Ii protein
Relatively little is known about the protein-protein interactions regulating the trafficking of the AT1 receptor (AT1R) through the biosynthetic pathway. Site-directed mutagenesis studies have defined the membrane-proximal region of the cytoplasmic tail of the AT1R as a site required for normal AT1R folding and surface expression. Based on yeast two-hybrid screening of a human embryonic kidney cDNA library with the AT1R carboxyl-terminal tail as a bait, we identified the Invariant chain (Ii) as a novel interacting protein. We confirmed the association by co-immunoprecipitation and co-localization studies. We localized the binding site for Ii on the AT1R carboxyl-terminal tail to a region that has been identified as important for exit of the AT1R from the endoplasmic reticulum (ER) and is conserved in many G protein–coupled receptors. Transient co-expression of Ii with the AT1R in CHO cells consistently reduced the AT1R density at the cell surface. Furthermore, the interaction of Ii with the carboxyl-terminal tail of the AT1R promotes the latter’s retention in the ER and its proteasomal degradation. Our observations indicate that Ii and the AT1R become associated in the early biosynthetic pathway and that the Ii protein is a negative regulator of AT1R expression.
Role of angiotensin II in prostate cancer
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 LNCaP, DU145, and PC3 prostate cancer cell lines. These 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 probably contributes to the growth and infiltration of malignant epithelial cells in the prostate. Furthermore, based on the recent observation that elevated levels of cytoplasmic Ang II can enhance cell proliferation via a non–AT1R mechanism, angiotensin-converting enzyme (ACE) inhibitors could also be of value in the treatment of prostate cancer by reducing intracellular Ang II formation.
Louis SN, Wang L, Chow L, Rezman 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 2007;31:391-5.
Caveolin and receptor signaling
Caveolin1 (Cav1) is an important component of plasma-membrane microdomains, such as caveolae and lipid rafts, that are associated with AT1 and EGF receptors in certain cell types. We analyzed the interactions between Cav1 and other signaling molecules that mediate AT1R function in Ang II- and EGF-stimulated hepatic C9 cells. Our analysis 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 in cells stimulated by EGF—and that Cav1 plays a scaffolding role in the process of MAPK activation. Furthermore, we found that intracellular Ca2+ and Src regulate Cav1 phosphorylation by Ang II and EGF. Phosphorylation of Cav1 and the EGFR by Ang II, but not ERK1/2 activation, is dependent on intracellular Ca2+. The PI 3-kinase inhibitors LY294002 and wortmannin differentially modulated both Cav1 and EGF receptor 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 are operative 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.
Angiotensin II and reactive oxygen species
We investigated the mechanism responsible for the Ang II–induced production of reactive oxygen species in non-phagocytic HEK293 cells and CHO cells reconstituted with the angiotensin type 1 receptor (AT1R), NADPH oxidase 1 (Nox1), Nox organizer 1 (Noxo1), and Nox activator 1 (Noxa1). Stimulation of the reconstituted cells with Ang II induced a substantially more superoxide production than the constitutive level mediated by the complex of Nox1, Noxo1, and Noxa1. The results demonstrate that Nox1 is activated by cell-surface receptor–mediated signaling and that the AT1R is coupled to Nox1. Expression of several AT1R mutants showed that interaction of the receptor with G proteins, but not with b-arrestin or with proteins (Jak2, phospholipase C-g1, and SHP2) that bind to the COOH-terminal region of the AT1R, was necessary for Ang II–induced superoxide production. Evaluation of the effects of constitutively active a subunits of G proteins and of various pharmacological agents suggested that signaling by a pathway comprising the AT1R, Gaq/11, phospholipase C-b, and protein kinase C was largely, but not exclusively, responsible for Ang II–induced activation of the Nox1-Noxo1-Noxa1 complex in the reconstituted cells. Our results also suggested a contribution of Ga12/13, phospholipase D, and phosphatidylinositol 3-kinase to Ang II–induced superoxide generation, whereas the small GTPase Rac1, Src, and the epidermal growth factor receptor did not appear to participate in this action of Ang II.
AT1 receptor structure and function
Using site-directed mutagenesis and molecular modeling, we investigated the network of inter-residue interactions within the transmembrane region of the AT1 receptor. Mutagenesis focused on residues Tyr292, Asn294, and Asn298 in transmembrane helix 7 and on the conserved Asp74 in helix 2 and other polar residues. By determining the effects of single and double-reciprocal mutations on agonist-induced AT1R activation, we evaluated functional interactions between pairs of residues. Reciprocal mutations of Asp74/Asn294 as well as of Ser115/Asn294, Ser252/Asn294, and Asn298/Ser115 caused additive impairment of function, suggesting that the pairs of residues make independent contributions to AT1R activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially augmented the impairment in inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded three-dimensional models of the transmembrane region of the AT1R and the mutants and of their complexes with Ang II, which we then used to identify possible mechanisms of impaired function of specific mutants. The data indicate that Asp74 and Asn298 are not optimally positioned for direct and strong interaction in the resting conformation of the AT1R. However, the balance of interactions between residues in helix 2 (such as D74) and helix 7 (such as N294, N295, and N298) of the AT1R is a crucial factor in determining the mutants’ functional activity and levels of expression.
Using molecular modeling, we evaluated the molecular mechanism of the constitutive activity of AT1R mutants at position 111. We found that the mechanism involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix 3 (TM3) from L112 to Y113 to F117, which in turn results in conformational changes in TM4 (residues I152 and M155) and leads to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis as well as with correct predictions of constitutive activity of mutants L112F and L112C.
Hunyady L, Catt KJ. Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol 2006;20:953-70.
Nikiforovich GV, Mihalik B, Catt KJ, Marshall GR. Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor. J Pept Res 2005;66:236-48.
Nikiforovich GV, Zhang M, Yang Q, Jagadeesh G, Chen H-C, Hunyady L, Marshall GR, Catt KJ. Interactions between conserved residues in transmembrane helices 2 and 7 during angiotensin AT1 receptor activation. Chem Biol Drug Des 2006;68:239-49.
Olivares-Reyes JA, Shah BH, Hernandez-Aranda J, Garcia-Caballero A, Farshori MP, Garcia-Sainz JA, Catt KJ. Agonist-induced interactions between angiotensin AT1 and epidermal growth factor receptors. Mol Pharmacol 2005;68:356-64.
1 Keiko Wada, MD, PhD, former Postdoctoral Fellow, now at Asahikawa Medical College Hospital, Asahikawa, Japan
COLLABORATOR
László Hunyady, MD, PhD, DSc, Semmelweis University of Medicine, Budapest, Hungary
Simon Louis, PhD, University of Melbourne, Austin Health, Heidelberg, Australia
William Louis, MD, University of Melbourne, Austin Health, Heidelberg, Australia
Garland R. Marshall, PhD, Washington University, St. Louis, MO
Nadia Mores, MD, Catholic University, Rome, Italy
Gregory Nikiforovich, PhD, DSc, Washington University, St. Louis, MO
J. Alberto Olivares-Reyes, PhD, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico
Sue Goo Rhee, PhD, Institute of Molecular Life Science and Technology, Ewha Women’s University, Seoul, South Korea
Bukhtiar Shah, DVM, PhD, Division of Physiology and Pathology, Center for Scientific Review, NIH, Bethesda, MD
Márta Szaszák, PhD, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
For further information, contact cattk@mail.nih.gov.
