Structure/Function Properties of the GnRH Receptor
Chung, Arora, Catt
The GnRH receptor is a unique tail-less member of the G protein-coupled receptor (GPCR) family. It signals primarily through the phosphoinositide/calcium pathway and activation of MAP kinases to promote secretory and growth responses. The GnRH receptor is coupled to Gq/11 via its second and third intracellular loops and to Gs via its first loop. The agonist-induced coupling of heptahelical receptors to their cognate G proteins often depends on the amino-terminal region of the third intracellular loop. Many GPCRs, including the GnRH and AT1 receptors, contain an apolar amino acid in this region at a constant distance from conserved Pro and Tyr/Asn residues in the fifth transmembrane domain. In the GnRH and angiotensin AT1 receptors, the residue (Leu237) is an important determinant of receptor expression and function. It is part of a conserved N/YxxIxxxL motif that is often present in the third intracellular loop of GPCRs and is likely to be of general importance in their expression, agonist-induced activation, coupling to cognate G proteins, and internalization.

The GnRH Pulse Generator
Krsmanovic, Martinez-Fuentes, Arora, Mores, and Chen in collaboration with Stojilkovic (SCS, ERRB)
The pulsatile secretory activity of GnRH-producing neurons in the hypothalamus, and consequently of pituitary gonadotroph cells, is essential for the maintenance of normal patterns of gonadotropin secretion and reproductive function. The process can be analyzed in vitro because the pulsatile release of GnRH also occurs in cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells). Both native and transformed GnRH neurons express GnRH receptors that mediate agonist- and antagonist-induced changes in episodic secretory activity. Studies on both hypothalamic cells and GT1-7 cells have shown that the feedback actions of GnRH on its receptors in the GnRH neuron are involved in the genesis of pulsatile neuropeptide secretion. Blockade of neuronal GnRH receptors by specific antagonists abolishes pulsatile secretion and causes a slowly progressive increase in GnRH release. These and other observations are consistent with the existence and function of autocrine regulation of the GnRH neuron by its neuropeptide product. Pituitary gonadotrophs also release GnRH as well as LH, and treatment with a GnRH receptor antagonist or GnRH antiserum reduces basal LH release. The presence and actions of GnRH in the anterior pituitary gland suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin secretion. In the hypothalamus, the regulatory actions of GnRH on cyclic AMP and calcium signaling, together with the ultrashort loop feedback effects of GnRH on its cells of origin, exert both positive and negative actions on neuropeptide release that serve to maintain the episodic mode of GnRH secretion.

Regulation of Calcium-Sensitive Adenylyl Cyclase in GnRH Neurons
Krsmanovic, Mores, Navarro, Tomic, Catt
GnRH production and secretion depend on both calcium and cyclic AMP levels in hypothalamic GnRH neurons. Studies on intracellular signaling in immortalized GnRH neurons revealed that cAMP production is elevated by increased extracellular Ca²⁺ and the Ca²⁺ channel agonist, BK-8644, and is diminished by low extracellular Ca²⁺ and treatment with nifedipine. These findings are consistent with the abundant expression of adenylyl cyclase type I (type I AC) in GnRH neurons. Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of Gi-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to Gi-related proteins at high levels of agonist activation. The stimulation of cAMP production by activation of endogenous luteinizing hormone receptors in GT1-7 cells is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca²⁺ entry through voltage-sensitive calcium channels, leading to activation of Ca²⁺-stimulated type I AC. Furthermore, the Ca²⁺ influx-dependent activation of AC I operates in conjunction with AC regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production in the GnRH neuron.

Regulatory Actions of Estrogen Receptors in GnRH Neurons
Navarro, Krsmanovic, Catt
We observed an interaction between estrogen receptors and intracellular signaling in hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells), both of which express estrogen (ERa and ERb) and progesterone receptors. Both cell types exhibited positive immunostaining for plasma membrane ERs as well as estradiol-induced changes in adenylyl cyclase activity. In GT1-7 cells, short-term treatment (five minutes) with estradiol caused dose-dependent inhibition of cAMP production. More prolonged treatment (60 minutes) with picomolar estradiol concentrations inhibited, while nanomolar concentrations increased, cAMP production. The ER antagonist ICI 182,780 abolished both inhibitory and stimulatory actions of estradiol on cAMP production. Estradiol-induced inhibition of adenylyl cyclase was also prevented by treatment with pertussis toxin, consistent with coupling of the membrane-bound estradiol receptors to an inhibitory G protein. Inhibitory actions of estradiol on adenylyl cyclase were also evident in membrane fractions and in cells treated with estrogen-protein conjugates. In perifused GT1-7 cells and hypothalamic neurons, treatment with ovulatory phase estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings have demonstrated that membrane-associated ER expressed in GnRH neurons interact with adenylyl cyclase inhibitory G proteins by a rapid nongenomic mechanism and modulate intracellular cAMP signaling and neuropeptide secretion.

Angiotensin Receptor Structure, Activation, and Phosphorylation
Hunyady, Olivares-Reyes, Smith, Catt
The goal of the project is to elucidate the molecular mechanisms of receptor activation and intracellular signaling that are initiated by the pressor octapeptide, angiotensin II (Ang II). Most of the diverse physiological actions of Ang II in cardiovascular, renal, neuronal, and other target cells are mediated by the Gq/11 protein-coupled AT1 receptor. In general, the functions of the distantly related AT2 receptor counteract the growth-related actions of the AT1 receptor. The third intracellular loop of the AT1 receptor and other GPCRs is an important determinant of G protein coupling. We previously identified four specific amino acids that are required for Gq/11 coupling to this domain of the AT1 receptor and probably to other GPCRs. They are the Tyr215 and Leu223 residues in the N-terminal region of the third intracellular loop and Ile238 and Phe239 in the N-terminal region. Agonist activation is accompanied by rapid phosphorylation of the AT1 receptor at several serine/threonine residues in its C-terminal intracellular tail and by sequestration of the agonist-receptor complex via a beta-arrestin- and clathrin-dependent endocytic mechanism. In contrast, the angiotensin AT2 receptor, which is coupled to activation of tyrosine phosphatase and inhibition of MAPK kinase, is phosphorylated at a single residue (Ser354) in its cytoplasmic tail and does not undergo agonist-induced internalization. In addition to rapid phosphorylation via PKC during homologous activation by Ang II, the AT2 receptor undergoes heterologous PKC-dependent phosphorylation during activation by the AT1 receptor. This process could contribute to activation of the counterregulatory action of AT2 receptors on AT1 receptor-mediated growth responses.

Properties of a Phosphorylation-Deficient AT1 Receptor
Olivares-Reyes, Smith, Hunyady, Shah, Catt
In most GPCRs, agonist binding is rapidly followed by phosphorylation of serine/threonine residues. It is predominantly mediated by specific receptor kinases (GRKs) and depends on adjacent acidic residues located in the C-terminal tail or the third intracellular loop of the receptor. No such residues are present in the AT1 receptor tail, but an analysis of the functional role of a diacidic motif (Asp236-Asp237) in its third intracellular loop revealed that substitution of both amino acids with alanine or asparagine residues diminished Ang II-induced AT1 receptor phosphorylation in COS-7 cells. However, the ability of the phosphorylation-deficient (DD-mutant) receptor to mediate Ang II-stimulated inositol phosphate production, MAP-kinase activation, and AT1 receptor desensitization and internalization was not significantly impaired. Overexpression of dominant negative GRK2(K220M) decreased agonist-induced receptor phosphorylation by about 40 percent but did not further reduce the impaired phosphorylation of the DD-mutant receptors. Inhibition of PKC by bisindolylmaleimide reduced the phosphorylation of both the wild-type and DD-mutant receptors by about 30 percent. The inhibitory effects of GRK2(K220M) expression and PKC inhibition on agonist-induced phosphorylation were additive for the wild-type AT1-R but not for the DD-mutant receptors. Agonist-induced internalization of the wild-type and DD-mutant receptors was similar and unaltered by coexpression of dominant negative GRK2. These findings indicate that an acidic motif in the third intracellular loop of the AT1-R is required for agonist-induced phosphorylation of its carboxyl-terminal tail by GRKs. The properties of the DD-mutant receptors suggest that Ang II-induced signaling as well as receptor desensitization and internalization do not depend on GRK-mediated phosphorylation of the agonist-activated AT1 receptor.

MAP Kinase Activation by the Angiotensin AT1 Receptor
Shah, 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. Agonist activation of endogenous AT1 receptors expressed in hepatic C9 cells markedly stimulates phosphoinositide hydrolysis and activates PKCd; the activation also stimulates phosphorylation of the proline-rich tyrosine kinase, Pyk-2, and activates ERK1/2. The stimulatory actions of Ang II on Pyk2 and ERK phosphorylation were abolished by PKC depletion and selective inhibition of PKCd by rottlerin, but not by Ca²⁺ chelators. These effects, and the similar actions of the Src kinase inhibition, implicate PKCd and Src kinase in ERK activation. Phorbol myristyl acetate (PMA) caused much greater phosphorylation of Pyk2 and ERK than ionomycin, and the effects of PMA and Ang II were abolished in PKC-depleted cells. Ang II also increased the association of Pyk2 with Src and with the EGF receptor (EGF-R). EGF caused much greater tyrosine phosphorylation of the EGF-R than Ang II and PMA. Ang II-induced activation of ERK but not of Pyk2 was prevented by inhibition of EGF receptor phosphorylation by AG1478 and of Src kinase by PP1. Ang II also increased the association of the adaptor protein, Grb2, with the EGF-R. These findings indicate that Src and Pyk2 act upstream of the EGF-R and that most of the Ang II-induced ERK phosphorylation depends on transactivation of the EGF-R. In summary, Ang II-induced ERK activation in C9 hepatic cells is initiated by a PKCd-dependent but Ca²⁺-independent mechanism and is mediated by the Src/Pyk2 complex through transactivation of the EGF-R.