We investigate the mechanisms by which peptide hormones control the activities of endocrine and other target cells. Major areas of interest include the characterization of receptors, signal transduction pathways, and other cellular processes involved in the neural control of gonadotropin-releasing hormone (GnRH) biosynthesis and secretion, the regulation and structure-function properties of receptors for GnRH and angiotensin II (Ang II), and the actions of GnRH and Ang II on target cells in the hypothalamus, pituitary, adrenal gland, and liver. Current research concerns the structural features and signal transduction pathways of the GnRH and Ang II receptors, and the manner in which these mechanisms regulate the secretory, metabolic, and growth responses of hypothalamic, pituitary, adrenal, and hepatic cells.

The GnRH Pulse Generator: Role of Agonist-Induced Switching of G Protein Signaling
Krsmanovic, Martinez-Fuentes, Navarro, Mores, Catt
The pulsatile secretory activity of the GnRH-producing neuronal network in the hypothalamus, and consequently of pituitary gonadotroph cells, is essential for the maintenance of normal patterns of gonadotropin secretion and reproductive function. Based on the capacity of cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells) for prolonged pulsatile release of GnRH, we have analyzed the cellular and biochemical mechanisms of episodic neurosecretion in vitro. Pulsatile GnRH secretion is highly calcium-dependent and is stimulated by increased cell excitability and cyclic AMP (cAMP). The GnRH secretory profile is also influenced by agonist activation of the endogenous GnRH receptor (GnRH-R), which couples to Gq/11, as indicated by reduction in membrane-bound aq/11 and increased inositol phosphate/Ca2+ signaling. In contrast, GnRH antagonists abolish pulsatile GnRH secretion and increase membrane-associated aq/11. GnRH stimulates cAMP production at low nanomolar concentrations but, at high concentrations, has an inhibitory effect that is abolished by pertussis toxin (PTX). Coupling of the GnRH-R to both Gs and Gi proteins was also indicated by the ability of nanomolar GnRH concentrations to reduce membrane-associated as and ai3 levels and of high concentrations to diminish ai3 levels. Conversely, ai3 was increased during GnRH antagonist and PTX treatment, with concomitant loss of pulsatile GnRH secretion. In cholera toxin (CTX)-treated GnRH neurons, decreases in as immunoreactivity and increases in cAMP production paralleled the responses to nanomolar GnRH concentrations. It is noteworthy that treatment with CTX and 8-bromo-cAMP amplifies episodic GnRH pulses but does not affect their frequency. These findings indicate that an agonist concentration–dependent switch in coupling of the GnRH-R between specific G proteins regulates Gq/11-InsP3/Ca2+ signaling as well as Gs-cAMP–stimulatory and Gi-cAMP–inhibitory responses. In a proposed model, this mechanism serves as a timer to regulate the frequency of Ca2+ - and cAMP-dependent episodes of GnRH release (Fig. 8).

Fig 8

A proposed mechanism of pulsatile GnRH secretion. GnRH neuronal firing promotes calcium influx, activation of adenylyl cyclase and cAMP production, and calcium signaling, which stimulate GnRH secretion. At high local GnRH concentrations, an autocrine switch from Gs to Gi interrupts the rise in GnRH release. This is followed by a fall to baseline and subsequent reactivation of neurosecretion by the resurgent calcium/cAMP signaling pathways. AC, adenylyl cyclase.


Regulatory Actions of Estrogen Receptors in GnRH Neurons
Navarro, Krsmanovic, Murdock, Catt
We have observed that hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells) express not only nuclear but also cell membrane receptors for estrogen receptors (ERa and ERb). Both cell types exhibit positive immunostaining for plasma-membrane ERs as well as estradiol-induced changes in adenylyl cyclase activity. In GT1-7 cells, physiological (picomolar) estradiol concentrations cause a dose-dependent inhibition of cAMP production that is abolished by the ER antagonist ICI 182,780. Estradiol-induced inhibition of adenylyl cyclase in cells and membranes is also prevented by treatment with PTX, consistent with coupling of the membrane-bound estradiol receptors to an inhibitory G protein. In perifused GT1-7 cells and hypothalamic neurons, treatment with ovulatory-phase estradiol levels prolongs the GnRH interpeak interval, shortens peak duration, and increases peak amplitude. These findings have demonstrated that the membrane-associated ER expressed in GnRH neurons exhibits high-affinity interactions with adenylyl cyclase inhibitory G proteins by means of a rapid non-genomic mechanism, and modulates intracellular cAMP signaling and neuropeptide secretion. Recent studies have indicated that the negative regulatory action of estradiol on cAMP production is associated with a direct interaction between ERa and the Gi a-subunit, as demon-strated by immunoprecipitation with a specific anti-ERa antibody. The agonist sensitivity of this interaction is commensurate with the low estradiol concentrations at which Gi-mediated inhibition of cAMP production is observed, suggesting that this process represents a physiological, negative feedback action of estrogen on the GnRH neuron.

Angiotensin Receptor Structure, Activation, and Phosphorylation
Olivares-Reyes, Shah, Catt; in collaboration with Hunyady, Balla
The AT1 angiotensin II (Ang II) receptor (AT1R) is a typical G protein-coupled receptor (GPCR) and mediates the known physiological actions of the pressor octapeptide hormone, angiotensin II. Many such actions are mediated by coupling of the AT1R to Gq/11 proteins and by activation of phosphoinositide-calcium signaling and phosphorylation cascades that regulate cell growth, differentiation, and function. Several of the latter actions of Ang II are mediated by transactivation of receptor tyrosine kinases, in particular the EGF receptor (EGF-R), followed by activation of ras-dependent stimulation of MAP kinases. In the C9 hepatic cell line, the large share of Ang II–induced ERK phosphoryla-tion depends on transactivation of the EGF-R, as described below. It has been proposed that the ability of certain GPCRs to activate MAP kinase depends on their agonist-induced phosphorylation, recruitment of arrestins, and clathrin-mediated endocytosis. However, studies on normal and mutant AT1 receptors expressed in C9 and COS cells revealed that Ang IIinduced ERK1/2 activation is independent of both AT1R and EGF-R endocytosis and is mediated by transactivation of the EGF-R. These and other studies have suggested that the dependence of MAP kinase activation on receptor endocytosis is confined to a minority of the GPCR family.

During agonist-induced endocytosis of the AT1R and many other GPCRs, invagination of clathrin-coated pits and vesicle formation depends on the recruitment of the 100 kDa GTPase, dynamin, and several other accessory proteins. An analysis of the roles of the functional domains of dynamin in endocytosis of the AT1-R revealed that, similar to the recruitment of dynamin-1 during recycling of synaptic vesicles, interaction of the proline-rich domain of dynamin-2 with SH3 domains of amphiphysins and endophilins is essential for agonist-induced internalization of the AT1 receptor. This mechanism could be of general importance in the dynamin-dependent endocyto-sis of other GPCRs in non-neural tissues. Related studies on the endocytosis and processing of the AT1-R that used a GFP-tagged receptor expressed in HEK cells identified two phases of the internalization and recycling of the receptor to the cell membrane. The internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies and return to the cell surface by a rapid, PI 3 kinase–dependent recycling pathway as well as by a slower pathway that is less sensitive to inhibition of PI 3-kinase.

Mechanisms of GPCR-Mediated Activation of MAP Kinase
Shah, Farshori, Catt
The ability of several agonist-activated GPCRs to stimulate MAP kinase activity and growth responses is mediated by a variety of intracellular pathways, including transactivation of growth factor receptors and their downstream signaling cascades to the nucleus. Our recent studies on agonist activation of endogenous AT1 receptors expressed in hepatic C9 cells revealed that angiotensin-stimulated phosphoinositide hydrolysis, activation of PKCd, and phosphorylation of the proline-rich tyrosine kinase Pyk2 were associated with phosphorylation and activation of ERK1/2. These and related findings have demonstrated that Ang II increases the association of Pyk2 with Src and with the EGF receptor (EGF-R) and that the majority of Ang II-induced ERK phosphorylation depends on transactivation of the EGF-R. In these hepatic cells, Ang IIinduced ERK activation is initiated by a PKCd-dependent but Ca2+-independent mechanism and is predominantly mediated by the Src/Pyk2 complex through transactivation of the EGF-R. 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 receptors expressed at the cell surface. Such studies are also in progress to evaluate the mechanisms and pathways that mediate the stimulation of MAP kinase responses in hypothalamic neuronal cells during activation of endogenous receptors for neurotransmitters, peptide hormones, and growth factors.