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PEPTIDE HORMONE RECEPTORS AND SIGNAL TRANSDUCTION
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Kevin J. Catt, MD, PhD, Head, Section on Hormonal Regulation |
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| Elucidation of the mechanisms by which peptide hormones control the cellular responses and signaling pathways of their endocrine and other target cells is the major focus of our research, particularly the characterization of the structure-function properties of specific G protein-coupled receptors (GPCRs) and their cellular processing and signal transduction pathways. We investigate the regulation and structure-function properties of the GPCRs for the central decapeptide reproductive hormone gonadotropin-releasing hormone (GnRH) and the vasoactive octapeptide angiotensin II (Ang II), and address the biochemical mechanisms that mediate their cellular actions. We perform our studies in normal and immortalized hypothalamic neurons, pituitary gonadotrophs, adrenal glomerulosa, and hepatic cells to elucidate the signaling pathways of the agonist-activated GnRH and Ang II receptors and the manner in which they regulate the metabolic, secretory, and growth responses of their respective target cells. Receptor-mediated regulation of the GnRH pulse generator Krsmanovic, Navarro, Martinez-Fuentes,b Catt; in collaboration with Mores The pulsatile secretory activity of the network of GnRH-producing neurons in the hypothalamus and, consequently, of the GnRH-regulated pituitary gonadotroph cells is essential for maintenance of the gonadotropin secretory profiles that ensure normal reproductive function. An analysis of the cellular and biochemical mechanisms of episodic neurosecretion in fetal hypothalamic cells and GT1-7 neuronal cells revealed that pulsatile GnRH secretion is calcium-dependent and regulated by cyclic AMP and cell excitability. It is also dependent on autocrine agonist activation of the endogenous GnRH receptor (GnRH-R) that we have identified in GnRH neurons and is abolished by GnRH antagonists. The agonist-activated GnRH-R couples primarily to Gq and to Gs and Gi proteins according to the prevailing GnRH concentration. These and other findings indicate that an agonist-induced concentration-dependent switch in coupling of the GnRH-R between specific G proteins regulates Gq/11-InsP3/Ca2+ signaling as well as Gs/cAMP-induced stimulatory and Gi/cAMP-dependent inhibitory responses. Our in vitro studies suggest that this autocrine inhibitory mechanism can function as a timer to regulate the frequency of Ca2+- and cAMP-dependent episodes of GnRH release. In related studies, we have found that hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells) express not only nuclear but also cell membrane receptors for the two estrogen receptor subtypes (ERalpha and ERbeta). The membrane-associated ERs expressed in GnRH neurons undergo high-affinity interactions with adenylyl cyclase- inhibitory G proteins, modulate intracellular cAMP signaling, and regulate the GnRH secretory profile. The sensitivity of this interaction to picomolar estradiol concentrations suggests that the process represents a physiological negative feedback action of estrogen on the GnRH neuron.
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. Navarro CE, Abdul Saeed S, Murdock C, Martinez-Fuentes AJ, Arora KK, Krsmanovic LZ, Catt KJ. Regulation of cyclic AMP signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized GnRH neurons. Mol Endocrinol 2003;17;1792-1804. Agonist-induced activation of MAP kinase in GnRH neuronal cells Shah, Farshori, Catt GPCRs mediate cellular responses to diverse extracellular messenger molecules. Recent studies have shown that GPCR-mediated signaling pathways include transactivation of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR), and receptors for platelet-derived growth factor, neurotrophins, and fibroblast growth factor. The complex cross-communication between GPCRs and RTKs affects sets of signaling molecules primarily determined by cell context and the types of receptors activated, which in turn elicits a variety of cellular effects during development, proliferation, differentiation, survival, repair, and synaptic transmission in the central nervous system. GnRH uses multiple signaling pathways to activate extracellularly regulated MAP kinases, such as ERK1/2, in normal and immortalized pituitary gonadotrophs and GnRH neurons (GT1-7 cells) and in transfected cells expressing the GnRH receptor. In GT1-7 cells, which express receptors for GnRH and epidermal growth factor (EGF), these agonists and phorbol myristate acetate (PMA) stimulate ERK1/2 phosphorylation. The actions of GnRH and PMA, but not that of EGF, depended on activation of protein kinase C (PKC) and on transactivation and phosphorylation of the EGF receptor. Both GnRH and EGF increased tyrosine-phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase Pyk2 at Tyr 402, reactions that depended on Ca2+ and activation of PKC. GnRH stimulation caused translocation of PKC alpha and epsilon to the cell membrane and enhanced the association of Src with Pyk2, EGFR, and the activated PKC isoforms. Inhibition of Src kinase and dominant negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA, but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGFR to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.
Related studies analyzed the mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation in non-neuronal HEK293 cells stably expressing the GnRH receptor (GnRHR). In this system, GnRH stimulation caused rapid and sustained phosphorylation of Pyk2 and ERK1/2 accompanied by their nuclear translocation. As expected, Pyk2 was also localized on cell membranes and focal adhesions. However, dominant regulatory Pyk2 had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. The actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of PKC and abolished by its inhibition. GnRH caused translocation of PKC alpha and delta isoforms to the cell membrane as well as phosphorylation of Raf at Ser 338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was mediated by activation of the EGFR receptor, whereas GnRH-induced ERK activation was completely independent of EGFR activation. The results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2 in HEK cells and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK cells is independent of Src and EGFR transactivation and mediated through the PKC/Raf/MEK cascade. The agonist-induced translocation of Pyk2 to the nucleus suggests that it may exert actions therein as well as at the periphery of the cell. The duration as well as magnitude of MAP kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. GnRH-induced activation of ERK1/2 by GnRH is transient in immortalized GT1-7 neurons but sustained in alphaT3-1 pituitary gonadotropes and GnRHR-transfected HEK293 cells. These cell types also expresses the EGFR and respond to EGF stimulation with marked and transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation due to EGFR transactivation was conned to GT1-7 cells, in which neither EGF nor GnRHR activation caused nuclear translocation of phospho-ERK1/2 into the nucleus. In contrast, agonist stimulation of GnRHRs expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a PKC-dependent but EGFR-independent pathway. These and other findings indicate that the duration of ERK1/2 activation depends on the signaling pathways used by GnRH in specific target cells and that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain GPCRs. Farshori PQ, Shah BH, Arora KK, Martinez-Fuentes A, Catt KJ. Activation and nuclear translocation of PKCdelta, Pyk2, and ERK1/2 by gonadotropin-releasing hormone in HEK293 cells. J Steroid Biochem Mol Biol 2003;85:337-347. Shah BH, Farshori MP, Jambusaria A, Catt KJ. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem 2003;278:19118-19126. Shah BH, Soh JW, Catt KJ. Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation. J Biol Chem 2003;278:2866-2875. Angiotensin AT1 receptor signaling Shah, Catt; in collaboration with Gaborik, Hunyady, Olivares Reyes The type 1 angiotensin II receptor (AT1R) mediates the known physiological actions of the octapeptide hormone angiotensin II (Ang II) on blood pressure regulation, aldosterone secretion, and sodium balance. Recently, in addition to its known involvement in arterial hypertension, Ang II activation of AT1 receptors in the cardiovascular system has been implicated in the development of atheroma and cardiac failure. Most of the actions of Ang II are mediated by coupling of the AT1R to Gq/11 proteins, followed rapidly by the initiation of phosphoinositide-calcium signaling and activation of PKC isoforms. These actions are associated with the onset of phosphorylation cascades to the nucleus and increased expression of genes regulating cell growth, differentiation, and function. Many of the growth-related actions of Ang II are mediated by transactivation of the EGF receptor (EGFR), which initiates ras-dependent stimulation of MAP kinase cascades. We have analyzed this process in C9 hepatic cells and AT1R-transfected COS-7 cells, in which Ang II-induced ERK phosphorylation proved to be largely dependent on transactivation of the EGFR and independent of the endocytosis of the AT1R and EGFR. In C9 cells, Ang II-induced ERK activation is initiated by a PKCdelta-dependent but Ca2+-independent mechanism and is predominantly mediated by the Src/Pyk2 complex through transactivation of the EGFR. Further investigations are addressing the nature of the interactions between GPCRs and receptor tyrosine kinases and the extent to which caveolae and other cell membrane structures are involved in the signaling cross-talk between different types of plasma-membrane receptors. In a mutational analysis of the function of a highly conserved domain (DRYXXV/IXXPL) in the second intracellular loop of the AT1R, individual Ala replacements of the Asp125 and Arg126, but not of Tyr127, moderately impaired agonist-induced inositol phosphate signaling. However, concomitant Asp/Arg substitutions markedly reduced both inositol phosphate signaling and AT1R internalization. Alanine-scanning showed that Ile130, His132, and Pro133 substitutions reduced agonist-induced inositol phosphate signal generation, whereas Met134 mutations also impaired receptor internalization. However, the D125A mutant AT1R exhibited moderate constitutive activity, as indicated by increased basal ERK activation and enhanced inositol phosphate responses to partial agonists. Agonist-induced stimulation of the Elk1 promoter showed parallel impairment with inositol phosphate signal generation after mutations in this region of the AT1R. The data suggest that Ca2+ signaling is required for the nuclear effects of angiotensin II-induced ERK activation, and are consistent with the role of the conserved DRY sequence in AT1R activation, and of Asp125 in constraining the receptor in its inactive conformation. It is likely that an apolar surface that includes Ile130 and Met134 in the cytoplasmic extension of the third transmembrane helix has a direct role in G protein coupling and signal generation. Gaborik Z, Jagadeesh G, Zhang M, Spat A, Catt KJ, Hunyady L. The role of a conserved region of the second intracellular loop in AT1 angiotensin receptor activation and signaling. Endocrinology 2003;144:2220-2228. Shah BH, Catt KJ. Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase Cdelta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor transactivation. Mol Pharmacol 2002;61:343-351. Shah BH, Catt KJ. A central role of EGF receptor transactivation in angiotensin II-induced cardiac hypertrophy. Trends Pharmacol Sci 2003;24:239-244. Angiotensin AT1 receptor endocytosis and cellular processing Shah, Baukal, Catt; in collaboration with Balla, Gaborik, Hunyady, Mihalika We previously demonstrated that agonist-induced endocytosis of AT1 receptors is a clathrin- and dynamin-dependent process at physiological Ang II concentrations but is independent of these proteins at high levels of agonist stimulation and that Gq-mediated receptor signaling at the membrane level, and to MAP kinase cascades, is independent of receptor endocytosis. Once internalized, Ang II-receptor complexes are processed via endosomes to structures that resemble multivesicular bodies and then return to the cell surface by a rapid recycling pathway that is dependent on PI 3-kinase. The receptor is also recycled via a slower pathway that is less sensitive to inhibition of PI 3-kinase. Related studies also found AT1 receptor endocytosis to be dependent on the interaction of the proline-rich domain of dynamin-2 with SH3 domains of amphiphysins and endophilins, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles.
In earlier studies, we observed that agonist-induced internalization of the AT1 receptor requires an STL triplet in its cytoplasmic C-terminal region and is associated with phosphorylation of a specific serine-/threonine-rich sequence in this domain. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor has been reported to regulate endocytosis of the receptor by facilitating its monoubiquitylation at lysine residues in its cytoplasmic tail. In our efforts to determine whether this process could contribute to the internalization of the AT1R, we relied on CHO cells to express mutant receptors with deletion or replacement of lysine residues in their agonist-sensitive serine/threonine-rich region. The modifications had no effect on the Ang II-induced receptor endocytosis, and fusion of ubiquitin in-frame to an internalization-deficient truncated AT1 receptor mutant did not restore the endocytosis of the resulting chimeric receptor. After substitution of all lysine residues in the serine/threonine-rich region, we observed no impairment of receptor internalization, when it was performed at saturating angiotensin II concentrations at which endocytosis occurs by a beta-arrestin- and dynamin-independent mechanism. These findings indicate that ubiquitylation of the AT1 receptor is not required for its agonist-induced internalization and suggest that endocytosis of mammalian GPCRs occurs by different mechanisms than those of yeast GPCRs. However, it is possible that ubiquitylation has other functions in the biosynthesis and trafficking of the AT1R and other GPCRs Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J Cell Biol 2002;157:1211-1222. Mihalik B, Gaborik Z, Varnai P, Clark AJ, Catt KJ, Hunyady L. Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region. Int J Biochem Cell Biol 2003;35:992-1002. Shah BH, Olivares-Reyes A, Yesilkaya A, Catt KJ. Independence of angiotensin II-induced MAP kinase activation from angiotensin type 1 receptor internalization in clone 9 hepatocytes. Mol Endocrinol 2002;16:610-620. Szaszak M, Gaborik Z, Turu G, McPherson PS, Clark AJ, Catt KJ, Hunyady L. Role of the proline- rich domain of dynamin-2 and its interactions with Src homology 3 domains during endocytosis of the AT1 angiotensin receptor. J Biol Chem 2002;277:21650-21656. COLLABORATORS Tamas Balla, MD, PhD, Endocrinology and Reproduction Research Branch, NICHD, Bethesda MD Richard Hauger, MD, University of California at San Diego, CA Laszlo Hunyady, MD, PhD, DSc, Semmelweis University of Medicine, Budapest, Hungary Nadia Mores, MD, Catholic University, Rome, Italy Alberto Olivares Reyes, PhD, National Polytechnical Institute and National University of Mexico, Mexico City, Mexico aBalázs Mihalik, PhD, formerly Semmelweis University of Medicine, Budapest, Hungary bAntonio Martinez-Fuentes, PhD, former Postdoctoral Fellow
For further information, contact catt@helix.nih.gov |
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