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  • 1. 51 The Gonadotropin-Releasing Hormone and Its Receptor L Jennes, University of Kentucky, Lexington, KY, USA A Ulloa-Aguirre, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico; Oregon National Primate Research Center, Beaverton, OR, USA J A Janovick, Oregon National Primate Research Center, Beaverton, OR, USA V V Adjan, University of Kentucky, Lexington, KY, USA P M Conn, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico; Oregon National Primate Research Center, Beaverton, OR, USA; Oregon Health and Science University, Portland, OR, USA ß 2009 Elsevier Inc. All rights reserved.Chapter Outline51.1 Introduction 164651.2 GnRH Neuronal Systems 164651.2.1 Embryonic Development of the GnRH System 164651.2.2 Postnatal and Adult GnRH Systems 164751.2.2.1 GnRH-containing cell bodies 164751.2.2.2 GnRH-containing projections 164751.2.2.3 Associations with the cerebrospinal fluid 164851.2.2.4 Cytology of GnRH hormone neurons 164851.2.3 Regulation of GnRH Neurons 164851.2.3.1 Catecholamines 164951.2.3.2 Glutamate 165051.2.3.3 Gamma-aminobutyric acid 165151.2.3.4 Neuropeptides 165251.3 GnRH Receptors in the Central Nervous System 165451.3.1 Localization of GnRH Receptors in the Brain 165451.3.2 Characterization of GnRH Receptors in the Brain 165451.3.3 Regulation of GnRH Receptor Expression in the Brain 165551.3.4 Functional Aspects of GnRH Receptors in the Brain 165651.4 Molecular and Cellular Mechanism of GnRH Action in the Anterior Pituitary 165651.4.1 GnRH Receptor 165651.4.2 Effector Coupling 165751.4.3 Receptor–Receptor Interactions 165751.4.4 Receptor Trafficking 165751.4.4.1 Endoplasmic reticulum quality control system and the role of endogenous chaperone proteins 165751.4.4.2 Mutant GnRHRs isolated from patients with hypogonadotropic hypogonadism are actually misfolded and misrouted proteins that can be rescued and restored to function 165851.4.4.3 The ability to rescue mutant proteins using pharmacoperones has therapeutic potential 165951.4.4.4 The rescue approach appears generally applicable to other mutant GPCRs, non-GPCR receptors, ion channels, and enzymes associated with disease: This supports the importance of understanding the mechanism of this event in a well-defined system 1659References 1660Further Reading 1668 1645
  • 2. 1646 The Gonadotropin-Releasing Hormone and Its Receptor51.1 Introduction most GnRH cells have reached their final destina- tion (Schwanzel-Fukuda and Pfaff, 1989; WrayGonadotropin-releasing hormone (GnRH) is a dec- et al., 1989a,b). However, many GnRH neuronsapeptide that is synthesized by relatively few neurons remain associated throughout life with the ganglionin the ventral forebrain. These neurons project axons terminale and the associate branches of the termi-to the median eminence of the mediobasal hypothal- nal nerve (Schwanzel-Fukuda and Silverman, 1980;amus where the hormone is released into the peri- Schwanzel-Fukuda and Pfaff, 1990, 1991; Jennes andvascular space of fenestrated capillaries and carried Schwanzel-Fukuda, 1992; Schwanzel-Fukuda et al.,through the blood to the anterior pituitary. GnRH 1992; Schwanzel-Fukuda, 1999; Jennes and Stumpf,binds to and activates specific membrane receptors 1980a). The time course of the migration of GnRHon the gonadotropes to stimulate the synthesis and neurons and initiation of GnRH peptide synthesisrelease of follicle-stimulating hormone (FSH) and has been confirmed with in situ hybridization studiesluteinizing hormone (LH). These pituitary hor- that showed that the neurons begin to synthesizemones, in turn, cause follicular growth and ovulation GnRH mRNA at E10 and that by E11 translationin the ovary, as well as steroid hormone synthesis. into GnRH peptide reaches detectable levels (WrayThe ovarian hormones, estrogen and progesterone, et al., 1989a). Additional experiments using thymi-feed back to the brain and pituitary in an inhibitory dine incorporation into newly formed DNA con-fashion throughout the reproductive cycle, except for firmed that GnRH neurons indeed originate froma very short period during proestrus when a change the olfactory placode and not from the ventricularto a positive feedback stimulates GnRH, LH, and lining (Schwanzel-Fukuda and Pfaff, 1989). ThisFSH release to induce ovulation. In the rat, the view is further supported by studies of the small-appropriate pulsatile release of GnRH is critical for eye mouse, which fails to develop eyes and olfactorythe maintenance of an estrous cycle and for repro- placodes due to a mutation in the pax-6 gene. Homo-duction in general. zygote mice do not contain GnRH neurons in the nasal region or in the forebrain, whereas hetero- zygotes contain a normal GnRH neuronal system51.2 GnRH Neuronal Systems (Dellovade et al., 1998). Several other regions in the mouse brain were described to contain neurons that51.2.1 Embryonic Development of the express GnRH transiently during the pre- and earlyGnRH System postnatal period, and it was shown that these neuronsAlthough GnRH neurons reside in the rostroventral in the lateral septum, bed nucleus of the stria term-forebrain in the adult mammal, these neurons origi- inals, and tectum are not derived from the olfactorynate in the olfactory placode. Using immunohisto- placode (Wu et al., 1995; Skynner et al., 1999). Thesechemistry, GnRH-synthesizing neurons are first cells synthesize the mammalian form of the GnRHidentified at embryonic day 11 (E11) in the epithe- decapeptide, as is suggested by their immunoreactiv-lium of the medial olfactory placode (Schwanzel- ity to a variety of well-characterized antibodies. TheFukuda and Pfaff, 1989; Wray et al., 1989a). Here, biological significance of this subset of GnRH neu-the GnRH neurons appear oval or fusiform and have rons is not understood; however, the lack of access ofnot yet developed identifiable axons (Zheng et al., these neurons to fenestrated capillaries precludes an1992). GnRH neurons begin to leave the epithelium endocrine E12 as cell clusters that migrate through the crib- The exact cell lineage of GnRH neurons is notriform plate to the ganglion terminale. During this clear. It was widely accepted that GnRH neuronsearly stage, approximately 50% of the GnRH neu- were closely related to olfactory sensory neuronsrons also express galanin and this percentage declines (Wray et al., 1989b), but later studies (Hilal et al.,as the migration continues (Key and Wray, 2000). 1996) showed that in chickens and in mice carrying aOver the subsequent few days, most GnRH cells mutation in the transcription factor activator proteinenter the ventromedial forebrain as part of the roots 2a (Kramer et al., 2000) GnRH neurons are present inof the nervous terminalis, and fewer cells reach the the respiratory epithelium. Moreover, the removal ofaccessory olfactory bulb along the vomeronasal the embryonic region of the nose that would differ-nerve. By E14, GnRH cells begin to reach the septum entiate into the respiratory epithelium eliminatesand preoptic region. The migration of the GnRH GnRH neurons, whereas the removal of the olfactoryneurons continues through birth and at that time epithelium region has no impact on GnRH
  • 3. The Gonadotropin-Releasing Hormone and Its Receptor 1647neuronal development (el Amraoui and Dubois, number of immunoreactive neurons rapidly declines1993). Together, these data indicate that GnRH pro- from rostral to caudal in the medial preoptic area andgenitor cells may actually be more closely related to anterior hypothalamic regions. Single GnRH neuronsrespiratory cells than to olfactory cells and more are consistently seen in a small region right above thestudies are needed to clarify the exact origin of supraoptic nuclei. In the rat, the mediobasal hypothal-GnRH neurons. amus does not contain immunoreactive GnRH Similarly, very little is known about the mechan- perikarya.isms that control the migration of GnRH neuronsfrom the nose into the forebrain. It appears that GnRH-containing projectionsstrands of GnRH neurons travel along a track that The most extensive axonal projection reaches thecontains the neural cell adhesion molecule (NCAM), median eminence of the ventral hypothalamus throughbut not laminin, cytotactin, fibronection, or cytactin diffuse projections that include periventricular, supra-(Schwanzel-Fukuda et al., 1992, 1994). These initial chiasmatic, and subchiasmatic pathways. Some of thesefindings stimulated a series of experiments in which projections traverse many hypothalamic nuclei, suchantibodies to NCAM were administered to 10-day- as the periventricular gray, paraventricular nucleus,old embryos, and it was shown that this treatment ventromedial nucleus, and arcuate nucleus. In thedisrupted the migration of embryonic GnRH neu- median eminence, GnRH-containing axons sproutrons (Schwanzel-Fukuda et al., 1994). However, extensively and most terminals are located in thebecause GnRH neurons reach their final position at external layer next to fenestrated capillaries, with thethe appropriate time in mice that do not express highest concentrations in the regions that sur-NCAM (Schwanzel-Fukuda et al., 1995), it appears round the infundibular sulcus (Silverman et al., 1979;that NCAM is not required for an appropriate migra- Merchenthaler et al., 1980, 1984; Jennes and Stumpf,tion (for review, see Schwanzel-Fukuda (1999)). 1980 a,b; Hoffman and Gibbs, 1982). This is the siteMigrating GnRH neurons follow a track of axons where GnRH is released into the portal blood to con-in vivo and in vitro that contain the intermediate trol the activity of the anterior pituitary gonadotropes.filament peripherin (Fueshko and Wray, 1994; Wray It has been shown in the rat that a single microin-et al., 1994). Peripherin is typically expressed in jection of retrograde tracer into the median eminenceolfactory receptor neurons (Gorham et al., 1991), labels approximately 65% of all GnRH neurons,and the close spatial relation between migrating which suggests that most GnRH neurons project toGnRH neurons and olfactory axons could indicate this neurohemal contact zone (Silverman et al., 1987).interactions of the two cell types in the guidance pro- Because such an injection covers only a small portioncess or common underlying mechanisms of guidance. of the median eminence, the percentage of GnRH neurons projecting to the median eminence is proba- bly substantially higher. An additional major GnRH-51.2.2 Postnatal and Adult GnRH Systems containing projection terminates in the OVLT, which like the median eminence is a circumventricularMany reviews have been published on the anat- organ that contains fenestrated capillaries. Althoughomy of the GnRH neuronal systems in the rodent it is clear that GnRH released in the median emi-(Barry et al., 1985; Jennes and Conn, 1994; Silverman nence controls LH and FSH release, the physiologi-et al., 1994); it is beyond the scope of this chapter cal significance of GnRH released at the OVLT is notto provide a detailed description of the entire known because only a venous vascular link exists withGnRH system. the pituitary portal system. The remaining projections of GnRH-containing51.2.2.1 GnRH-containing cell bodies axons are rather limited and involve only a few axonsIn the postnatal rat, most GnRH neurons are located per site. Areas in the brain that contain GnRH axonsaround the organum vasculosum of the lamina termi- include olfactory structures, such as the main andnalis (OVLT) in a dispersed distribution pattern that accessory olfactory bulb, the islands of Calleja, andis reminiscent of an inverted Y. GnRH neurons the pyramidal region of the piriform cortex. In addi-extend from the medial portions of the horizontal tion, a few axons reach the medial and cortical nucleilimb of the diagonal band through the vertical limb of the amgdala via the ventral amygdalal–fugal path-into the medial septum. Slightly fewer neurons are way and the stria teminalis. Single axons are also seenpresent in the rostral periventricular area, and the in the ventral hippocampus and the subiculum.
  • 4. 1648 The Gonadotropin-Releasing Hormone and Its ReceptorCaudal projections pass from the medial septum– Cytology of GnRH hormone neuronsdiagonal band through the stria medullaris, medial GnRH neurons appear as unipolar or bipolar cellshabenula, and the fasciculus retroflexus to the inter- that can have either smooth contours or have a spinypeduncular nucleus from which single fibers turn appearance; however, the significance of these mor-dorsally into the raphe nuclei and the central gray. phological differences has not been elucidated (Krisch, 1980; Liposits et al., 1984; Jennes et al., Associations with the 1985; Wray and Hoffman, 1986). Ultrastructural stud-cerebrospinal fluid ies have shown that GnRH neurons are rather simpleA unique feature of the GnRH neuronal system that neurons that have all the standard organelles requiredmay have important functional implications is the for appropriate functioning (Mazzuca, 1977; Jennesclose anatomical association of GnRH axons to et al., 1985; Witkin and Demasio, 1990). While earlierthe ventricular and subarachnoid cerebrospinal fluid light and electron microscopic immunohistochemical(Burchanowski et al., 1979; Jennes and Stumpf, studies have failed to identify dendrites of GnRH1980b). Thus, GnRH fibers are present consistently neurons ( Jennes et al., 1985; Witkin and Silverman,in or next to the ependyma of the ventromedial 1985), more recent studies using microinjections ofportion of the septal lateral ventricles, the ventral biocytin into green fluorescent protein expressingportions of the hypothalamic third ventricle, the sub- GnRH neurons clearly show that GnRH neuronsfornical organ, and the aqueduct. It appears that, at have long, mostly unbranched dendrites which con-least in some of these regions, GnRH axons penetrate tain many spine-like extensions (Campbell et al.,the ependyma and thus have direct contact with the 2005). GnRH axons form presynaptic specializationsinternal cerebrospinal fluid. Similar contacts occur on the dendrites or perikarya of other hypothalamicon the outer surface of the brain, especially at the neurons, which indicates that the GnRH peptideolfactory bulb where the ganglion terminale and the is released at these sites and probably acts as anervus terminalis reside in the subarachnoid space, neurotransmitter ( Jennes et al., 1985; Witkin andthe prechiasmatic cistern, and the interpeduncular Silverman, 1985). Interestingly, some of the postsyn-cistern. Based on these anatomical findings and stud- aptic neurons also contain GnRH, and it has beenies that measured GnRH levels in the cerebrospinal suggested that this kind of innervation could representfluid (Van Vugt et al., 1985; Skinner et al., 1995), it a mechanism by which the activity of GnRH neuronalis suggested that release of the hormone into the system is synchronized (Leranth et al., 1985b; Witkinportal blood may be coupled or synchronized with and Silverman, 1985). However, such contacts betweenthe release of GnRH into the cerebrospinal fluid. GnRH neurons are fairly rare and may not be suffi-This view is supported by the finding that increased ciently frequent to represent a relevant mechanism oflevels of GnRH and LH in the blood are paralleled activity coupling among the GnRH increases in the GnRH concentrations in thecerebrospinal fluid. GnRH in the cerebrospinal 51.2.3 Regulation of GnRH Neuronsfluid could reach distant sites of action and haveprolonged effects because endo- or exopeptidases The protein synthesis and release activity of GnRHare present only in very low levels in these fluid- neurons is regulated by complex feedback loops thatfilled compartments (Mendez et al., 1990). The sce- include the gonadal steroid hormones estradiol andnario of a coordinated release of GnRH could repre- progesterone, as well as a large number of neuro-sent an efficient way by which the endocrine effects transmitter systems (for review, see Kalra (1993),of GnRH on the pituitary gonadotropes could be Kordon et al. (1994), and Levine (1997)). Estradiolcoupled with the intracerebral effects of GnRH, for exhibits an inhibitory effect on GnRH neuronal sys-example, the facilitation of reproductive behaviors. tems during all phases of the reproductive cycle,An additional mechanism by which GnRH release at except for the proestrous stage when rising estradioltwo anatomically distinct sites could be coordinated levels cause a switch to a positive feedback mode thathas been suggested based on multiple retrograde stimulates GnRH release, which in turn causes a mas-labeling experiments that show that one GnRH neu- sive surge in pituitary LH secretion that is necessaryron can terminate at the perivascular space of fene- for ovulation. The mechanisms underlying this tempo-strated capillaries and, through axon collaterals, at rary positive feedback are not known but it appearsspecific nuclei in the brain, such as the interpedun- that direct effects of estradiol on GnRH neurons arecular nucleus ( Jennes, 1991). probably not the trigger for the LH surge. GnRH
  • 5. The Gonadotropin-Releasing Hormone and Its Receptor 1649neurons express only low levels of estrogen receptor- b (Hoffman, 1985). Although the limitations of the(ER-b) which is not necessary for the induction of resolution of light microscopy do not permit a distinc-ovulation (Hrabovszky et al., 2000). However, the man- tion between synaptic contacts and en passant axons,datory ER-a is not present in GnRH neurons. It is a close proximity of axons to GnRH neurons pro-therefore thought that input from steroid-sensitive vides an anatomical rationale for further in-depthinterneurons to the GnRH neurons is required for studies. Additional electron microscopic studies havethe generation of a GnRH-mediated LH surge. determined that some of these close appositions are Many studies have focused on the identification indeed synaptic in nature and that tyrosine hydroxy-of neurotransmitter systems that regulate GnRH lase (Leranth et al., 1988a; Chen et al., 1989b), gamma-neuronal activity by administering agonists or antag- aminobutyric acid (GABA) (Leranth et al., 1988b),onists during the preovulatory or estrogen- and glutamate (Goldsmith et al., 1994), or b-endorphinprogesterone-induced LH surge. Data from such (Chen et al., 1989a) are contained in these terminals.studies are summarized in several excellent reviews(Kalra, 1993; Kordon et al., 1994; Levine, 1997). Col- Catecholamineslectively, these studies show that most manipulations Noradrenergic and adrenergic axons are in closeof a neurotransmitter system have an impact on proximity to GnRH neurons (Moore et al., 1999).GnRH-mediated LH release, either stimulating LH Some of these fibers form synaptic complexessecretion to various degrees or reducing circulating with the GnRH neurons (Leranth et al., 1988b;LH levels. Most treatments that result in a stimulation Chen et al., 1989b), and GnRH neurons express theof GnRH neurons produce a two- to fivefold increase a1B adrenergic receptor mRNA (Petersen et al.,in LH levels, which is substantially smaller than LH 1999) and protein (Hosny and Jennes, 1998). Thislevels during the surge. So far, only a sequential treat- suggests that the catecholamines participate directlyment with estrogen and progesterone has been able in the control of GnRH neuronal activity. Oneto induce proestrus-like LH levels. On the other requirement for an involvement in the regulation ofhand, most antagonists of stimulatory neurotrans- GnRH neurons is the capability of the catecholamin-mitters can prevent the surge. These findings suggest ergic neurons to convey the steroidal signal. Severalthat it is probably not a single neurotransmitter sys- studies have provided strong evidence that this istem that induces GnRH neurons to release preovula- indeed the case. Thus, noradrenergic neurons accu-tory amounts of the peptide but instead several mulate 3H-estradiol in their nuclei (Heritage et al.,systems that participate either in parallel or sequen- 1977, 1980), ER-a, and probably ER-b; mRNA istially in the induction of the surge. Interference with present in the appropriate regions A1 and A2 of theonly one of these stimulators abolishes the surge. brainstem (Shughrue et al., 1997); and many of the One approach to identifying the neurotransmitters noradrenergic and adrenergic neurons contain ER-aand neuropeptides that participate in the control of protein (Simonian and Herbison, 1997; Lee et al.,GnRH neuronal activity is to use multiple immuno- 2000a). Moreover, tyrosine hydroxylase (Liaw et al.,histochemical stainings for GnRH and select neuro- 1992) and dopamine-b-hydroxylase gene expressiontransmitter, to determine which transmitter is present (Serova et al., 2000) are stimulated by estradiol, andin axons that are next to GnRH cell bodies. These the noradrenergic and adrenergic neurons in thestudies have demonstrated that catecholaminergic brainstem respond to estradiol by transient synthesis(Ajika, 1979; Jennes et al., 1982, 1983; Hoffman et al., of the transcription factor fos (Lee et al., 2000a). The1982; Leranth et al., 1988a; Chen et al., 1989b), sero- noradrenergic and adrenergic input to the GnRHtoninergic ( Jennes et al., 1982; Kiss and Halasz, 1985), system is critical for the maintenance of pulsatilegamma-aminobutyric acidergic (GABAergic; Jennes GnRH release and the induction of a preovulatoryet al., 1983; Leranth et al., 1985a; Witkin, 1992), and and estrogen- and progesterone-induced LH surge,glutamatergic axons (Goldsmith et al., 1994; Eyigor as has been shown repeatedly with physiologicaland Jennes, 1997) were juxtaposed to GnRH neurons approaches (for review, see Kordon et al. (1994) andas were axons that contained the neuropeptides, Herbison (1997a)). Thus, the inhibition of eitherb-endorphin (Leranth et al., 1988a; Chen et al., norepinephrine or epinephrine synthesis results1989a,b), galanin (Merchenthaler et al., 1991), neuro- in a markedly reduced LH secretion in ovariecto-peptide Y (Li et al., 1999), vasoactive intestinal peptide mized rats, suggesting that both neurotransmitters(VIP; van der Beek et al., 1993, 1994; Smith et al., regulate basal pulsatile GnRH–LH release (Drouva2000), substance P (Hoffman, 1985), or neurotensin and Gallo, 1976). Similar treatments with specific
  • 6. 1650 The Gonadotropin-Releasing Hormone and Its Receptorsynthesis inhibitors also prevent the steroid-induced GlutamateLH surge, indicating an important role in the genera- Immunohistochemical studies have shown that glu-tion of the preovulatory surge (Kalra et al., 1972; tamate or neuron-specific vesicular glutamate trans-Kalra and McCann, 1974; Crowley and Terry, 1981; porters (VGLUTs) are present in many axonCrowley et al., 1982; Coen and Coombs, 1983; for terminals that innervate GnRH neurons (Goldsmithreview, see Herbison (1997a)). On the other hand, et al., 1994; Lin et al., 2003), which suggests an impor-the administration of norepinephrine, epinephrine, tant stimulatory role of this excitatory neurotransmit-or a-adrenergic agonists causes a significant elevation ter in the control of GnRH neurons (Figure 1). Theof GnRH-stimulated LH levels (Rubinstein and findings that glutamatergic neurons in the septalSawyer, 1970; Gallo and Drouva, 1979; Kalra and complex and the hypothalamic arcuate and ventro-Gallo, 1983; Barraclough et al., 1984; Levine et al., medial nuclei as well as in the preoptic region express1991). These pharmacological studies are of physio- ER-a (Thind and Goldsmith, 1997; Eyigor et al.,logical relevance because measurements of norepi- 2004) and that short-term treatment with estradiolnephrine and epinephrine release (Mohankumar causes an increase in glutamate content (Luine et al.,et al., 1994) and turnover during select phases of the 1997) suggest that the glutamate neurons are affectedestrous cycle or the steroid-induced LH surge show by gonadal steroids and could convey the steroidalthat catecholamine turnover in the median eminence signals to the GnRH neurons. Glutamate release inis increased just before and during the LH surge the preoptic region is increased just prior to and(Wise et al., 1981; Wise, 1982; Adler et al., 1983; during the steroid-induced LH surge, as measuredSheaves et al., 1984). Together, based on anatomical with push–pull perfusion or in vivo dialysis ( Jarryand physiological evidence, the catecholamines can et al., 1992, 1995; Ping et al., 1994; Demling et al.,be viewed as important participants in the regulation 1985). Many studies have shown that glutamate andof GnRH release. its agonists AMPA, N-methyl-D-aspartate (NMDA), (a) (d) VGLUT2 (b) Synaptophysin (c) GnRH Overlay 20 µmFigure 1 Example of immunohistochemical triple labeling for (a) VGLUT2, (b) synaptophysin, and (c) GnRH as well as (d)an overlay of the three images, showing that many glutamatergic terminals are juxtaposed to the GnRH neuron, and someof these terminals contain synaptophysin (arrowheads). Reproduced from Lin W, McKinney K, Liu L, Lakhlani S, and Jennes L(2003) Distribution of vesicular glutamate transporter-2 messenger ribonucleic acid and protein in the septum-hypothalamusof the rat. Endocrinology 144(2): 662–670, Copyright 2003, The Endocrine Society.
  • 7. The Gonadotropin-Releasing Hormone and Its Receptor 1651 GnRH GluR1 Fos OverlayFigure 2 Example of triple-label immunohistochemistry for GnRH (green), AMPA receptor subunit GluR1 (red) and fosprotein (blue), as well as an overlay of the three images. Scale bar ¼ 30 mm. Reproduced from Bailey JD, Centers A, andJennes L (2006) Expression of AMPA receptor subunits (GluR1-GluR4) in gonadotrophin releasing hormone neurons of youngand middle-aged persistently oestrous rats during the steroid-induced luteinising hormone surge. Journal ofNeuroendocrinology 18(1): 1–12, with permission from Blackwell Publishing.and kainate (KA) stimulate GnRH–LH release in vivo indicates a preferential activation of KA2-containingand in vitro in a dose-dependent manner and, con- GnRH neurons (Eyigor and Jennes, 2000). Similarly,versely, that the blockade of either glutamate receptor the expression of the AMPA subunits GluR1 andsubtype with specific antagonists prevents the surge GluR3 increases significantly in GnRH neurons at(for review, see Brann and Mahesh (1997)). the time just before and during the LH surge, again In order to identify some of the mechanisms by preferentially in GnRH neurons that are activated aswhich glutamate regulates GnRH neurons, GnRH determined by the presence of fos (Bailey et al., 2006).neurons were screened for the presence of various However, glutamate or glutamate agonist administra-glutamate receptor subunit mRNAs and proteins. tion alone produces only a modest increase inThese studies showed that GnRH neurons express GnRH–LH release, and the coordinated release ofmost, if not all, ionotropic glutamate receptor sub- other transmitters is needed to induce a full surge.units (Eyigor and Jennes, 1997; Gore et al., 1996;Ottem and Petersen, 2000; Miller and Gore, 2002; Gamma-aminobutyric acidBailey et al., 2006). Examples are shown in Figure 2. GABA is the major inhibitory neurotransmitter in theSeveral lines of evidence suggest that these glutamate brain and strong evidence exists for an important inhib-receptors are indeed activated during the steroid- itory role in the control of GnRH neuronal activity.induced LH surge. For instance, approximately Thus, intracerebral microinjections of GABA into the50–60% of the GnRH neurons that express transiently preoptic area inhibit the proestrous surge (Herbisonthe transcription factor fos during the LH surge also and Dyer, 1991), and the administration of GABAA andexpress KA2 and GluR5 receptor subunits, which GABAB receptor antagonists has only modest effects
  • 8. 1652 The Gonadotropin-Releasing Hormone and Its Receptoron basal LH release; however, they greatly potentiate membrane; however, a definitive characterization ofthe stimulatory effects of norepinephrine (Hartman such receptors has not been al., 1990). Extracellular GABA concentrations, asmeasured with push–pull perfusion approaches, Neuropeptidesdecline dramatically prior to and during the estrogen- There are many neuropeptides that have been showninduced LH surge, which suggests that the release of a to participate in the regulation of GnRH releasetonic inhibition of the rostral hypothalamic neurons by (for review, see Kordon et al. (1994)); it is beyondGABA parallels the secretory activity of GnRH neu- the scope of this chapter to discuss the effects of allrons ( Jarry et al., 1995). Such a release of a continuous these peptides. We focus here on neuropeptide Yinhibition may also be an important mechanism that (NPY), vasoactive intestinal polypeptide (VIP),participates in the induction of puberty. Thus, GABA b-endorphin, and kisspeptins, because we knowlevels are very high in the mediobasal hypothalamus of most about their actions and receptor expression.the prepubertal monkey and decrease during early andmidpuberty, which is the exact opposite of GnRH 51.2.3(i) Neuropeptide Ylevels (Terasawa et al., 1999). GABAergic neurons con- NPY is probably the best-understood neuropeptidecentrate 3H-estradiol in their nuclei (Flugge et al., ¨ that is part of an excitatory circuit enhancing GnRH1986); contain ER-a protein in their nuclei (Herbison release. Several studies have shown that the adminis-et al., 1993); have extracellular GABA concentrations tration of NPY stimulates GnRH release (Crowleymodulated by estrogen (for review, see Herbison et al., 1987; Crowley and Kalra, 1987; Sabatino et al.,(1997b)); and are, from an anatomical point of view, 1989), whereas intraventricular injection of specificlocated in the appropriate hypothalamic areas to inner- anti-NPY antibodies (Wehrenberg et al., 1989) orvate GnRH neurons. That GABA can control GnRH antisense oligonucleotides (Kalra et al., 1995) blocksneurons directly at the perikaryon or axon terminal is the steroid-induced LH surge. These data suggestsuggested by several studies. Thus, glutamic acid that endogenous NPY is required for the occurrencedecarboxylase (GAD), the rate-limiting enzyme in of a surge. The observations that NPY mRNA con-GABA synthesis, is present in many axons that sur- tent in the arcuate nucleus increases prior to theround GnRH neurons ( Jennes et al., 1983), and Ler- onset of an LH surge (Bauer-Dantoin et al., 1992;anth et al. (1985a) have shown with electron Sahu et al., 1994), that NPY peptide levels in themicroscopy that GAD immunoreactive axons form mediobasal hypothalamus parallel LH levels (Sahupresynaptic terminals on GnRH perikarya. Evidence et al., 1989), and NPY release is enhanced during thethat these synapses are functioning is indicated by the LH surge (Watanobe and Takebe, 1992) supportobservation that approximately 75% of the GnRH the view that the NPY neurons participate in theneurons express the b-subunit of GABAA receptors, stimulation of GnRH release.whereas the a1- or b2-subunit mRNAwas not detected The NPY neurons that are involved in the control(Petersen et al., 1993). Later studies found that approx- of GnRH neurons are located in the brainstem,imately 25% of the GnRH neurons express the a1- or where NPY is expressed in catecholaminergic neu-a2-subunit mRNA, whereas only a small percentage rons (Everitt et al., 1984) and in the arcuate nucleusof the GnRH neurons contain detectable levels of (Chronwall et al., 1985; McShane et al., 1994). Both ofthe a1- and a2-subunit protein ( Jung et al., 1998). these neuronal cell groups express receptors forThe same study detected b3-subunit mRNA in only estrogen (Sar et al., 1990), and could therefore trans-approximately 25% of the GnRH neurons; g1 mRNA mit the steroid signal to the GnRH neurons. Thewas not detected. These receptor subunits can form NPY neurons project to the median eminence,functioning receptors as determined by electrophysio- among others, where NPY is released into the peri-logical measurements of membrane patches isolated vascular space for transport to the anterior pituitaryfrom identified GnRH neurons. These studies showed or for interactions with the axon terminals of otherthat all GnRH neuronal membrane patches responded neurons. In addition, NPY-containing axons reachto GABA and that the responses were inhibited by the the medial sepum–diagonal band–rostral preopticGABAA antagonist bicuculline (Spergel et al., 1999). area where the GnRH neurons are located. EvidenceTogether, these studies show that certain GABAA for direct actions on the GnRH neurons was providedreceptor subunits are present in GnRH neurons and, by electron microscopic studies that identified NPYbased on the subunit composition, that they could in presynaptic specialization on GnRH perikaryaform functioning GABA receptors in the plasma (Tillet et al., 1989; Tsuruo et al., 1990). Because
  • 9. The Gonadotropin-Releasing Hormone and Its Receptor 1653NPY can bind to and activate several different the VIP-2 receptor and that VIP immunoreactivemembrane receptor subtypes, it was important to axons were present next to most of the GnRH anddetermine which subtype is expressed in GnRH neu- VIP-2 receptor positive neurons (Smith et al., 2000).rons. Previous experiments have suggested that the Together, these data suggest that VIP neurons from theactivation of the NPY-Y1 receptor subtype is suprachiasmatic nucleus participate directly in therequired for the occurrence of a steroid-induced control of GnRH neurons through VIP-2 receptors.LH surge (Kalra et al., 1992) and a preovulatory LHsurge (Leupen et al., 1997), and it was shown that Endogenous opiatesNPY-Y1 receptor expression in the hypothalamus is The endogenous opiates include three families ofgreatly elevated just prior to and during the proes- neuropeptides (the endorphins, dinorphins, and enke-trous LH surge (Xu et al., 2000). Immunohistochemi- phalins), all of which have inhibitory effects on bothcal studies have confirmed that the NPY-Y1 receptor the frequency and amplitude of GnRH-mediatedprotein is present in many GnRH nerve terminals in LH release pulses (Bruni et al., 1977; Gilbeau et al.,the median eminence; however, the protein was 1985; for review, see Kalra (1993) and Kordon et al.not detected in GnRH perikarya (Li et al., 1999). (1994)). Conversely, the administration of the generalThese findings suggest that although the NPY-Y1 opiate-receptor blocker naloxone (Bruni et al., 1977;receptor subtype probably mediates the stimulatory Van Vugt et al., 1982) or the immunoneutralization ofeffects of NPY on GnRH release in the median b-endorphin (Weesner and Malven, 1990) causes aneminence, the effects of NPY on GnRH perikarya increase in circulating LH levels, which suggests thatmay involve another NPY receptor subtype that has endogenous endorphin is an important neurotrans-not been identified. mitter that tonically inhibits the GnRH neurons. This view is supported by studies that measured decreasing51.2.3(ii) Vasoactive intestinal polypeptide b-endorphin levels in the portal vessels on the after-It is clear now that the preovulatory LH surge is noon of proestrus just prior to the onset of the LHgenerated by a coupling of the steroidal signals with surge (Sarkar and Yen, 1985), indicating that thecircadian signals that originate from the suprachias- release of the GnRH neuronal system from the tonicmatic nucleus. In this nucleus, VIP is synthesized in inhibition is a physiologically relevant event and not aa large number of neurons that are located in the pharmacological artifact.ventrolateral aspects of the nucleus. Axons from these Endorphin-containing perikarya are restricted toVIP neurons project to GnRH neurons, among the hypothalamic arcuate nucleus and a considerableothers, where close appositions have been described. number of the endorphin-positive neurons alsoInterestingly, such close contacts occur with the sub- express estrogen receptors ( Jirikowski et al., 1986),population of GnRH neurons that express fos during suggesting that this neuronal system is a strong can-the steroid-induced LH surge, which suggests that didate to propagate the estrogen signal to other neu-VIP-containing axons exert a stimulatory effect rons. From the perikarya in the arcuate nucleus,on the GnRH neurons (van der Beek et al., 1994). extensive projections reach the median eminence,Although the physiological effects of VIP on GnRH preoptic area, and medial septum–diagonal bandrelease have not been fully elucidated and are, in part, (Khatchaturian et al., 1985), among others, whichcontroversial, several lines of evidence support are the areas in which a direct innervation of thethe view that VIP stimulates GnRH release. Thus, GnRH neuronal system could occur. Indeed, presyn-the central administration of VIP antiserum or aptic b-endorphin-containing specializations onadministration of VIP antisense oligonucleotides to GnRH perikarya have been described (Leranth et al.,the suprachiasmatic nucleus delays and reduces the 1988a; Chen et al., 1989a). However, several studiesmagnitude of the steroid-induced GnRH–LH surge were unable to detect the mRNA for any of the opiate(van der Beek et al., 1999; Harney et al., 1996) and receptor subtypes (Mitchell et al., 1997; Sannella andlesions of the suprachiasmatic nucleus abolish the Petersen, 1997), and it remains to be determined ifproestrous surge (Gray et al., 1978). In order to deter- these negative data are due to limited sensitivities ofmine whether VIP acts directly on GnRH neurons, dual in situ hybridization procedures or if, indeed,immunohistochemical triple-labeling experiments GnRH neurons do not synthesize opioid receptors.were conducted using antibodies to GnRH, VIP, and A large body of evidence suggests that the opioidthe VIP-2 receptor. The results of these studies show system interacts with other neurotransmitter systems,that approximately 40% of the GnRH neurons express which in turn could influence GnRH neuronal activity
  • 10. 1654 The Gonadotropin-Releasing Hormone and Its Receptor(for review, see Kalra (1993)); the interactions with 51.3 GnRH Receptors in the Centralthe catecholaminergic and NPY-containing neurons Nervous Systemespecially warrant further detailed investigations. 51.3.1 Localization of GnRH Receptors in51.2.3(iv) Kisspeptins the BrainRecently, a novel peptide has been identified to be The finding that GnRH is present in presynaptica very potent stimulator of GnRH release. This terminals and in axons that project to regions in thepeptide which is derived from the Kiss1 gene is brain that are not related to the direct regulation ofsynthesized as a 145-amino-acid long peptide that is the anterior pituitary gonadotrope function and stud-further processed to a 54-, 14-, 13-, and 10-amino- ies that showed a facilitatory effect of exogenousacid long peptide (Ohtaki et al., 2001). Kisspeptin is GnRH on reproductive behaviors prompted thesynthesized in neurons of the arcuate and the search for intracerebral receptors for GnRH. Initially,anteroventral periventricular nuclei, among others, in vitro autoradiography was used to localize thewhich are critical sites in the brain involved in the GnRH binding sites in the brain, followed by bio-control of GnRH neuronal activity (Gottsch et al., chemical assays that determined the binding charac-2004; Brailoiu et al., 2005). The findings that teristics of GnRH (Badr and Pelletier, 1987; Reubikisspeptin-producing neurons in the hypothalamus et al., 1987; Jennes et al., 1988; Leblanc et al., 1988).express estrogen receptors and kisspeptin mRNA These studies showed that specific binding sites forlevels are regulated by gonadal steroids support the GnRH exist in many regions of the central nervousview that this peptide is important for the control of system that had been implicated either in the inte-GnRH neurons (Smith et al., 2005a,b). While a direct, gration of olfactory and vomeronasal cues or in sitessynaptic innervation of GnRH neurons by kisspeptin- that are known to be involved in the generation ofcontaining axon terminals has not yet been clearly emotional and reproductive behaviors. Thus, bindingestablished, many data support the view that they sites for GnRH are present in the laminae glomer-do exist. Thus, kisspeptin-54 and kisspeptin-10 are ulosa and plexiformis of the olfactory bulb, theextraordinarily potent in causing GnRH release, and nucleus olfactorius anterior, the frontal cortex at theintracerebroventricular administration of only 1-fmol sulcus rhinalis, and the piriform cortex. In the sep-(Gottsch et al., 2004) or 10-pmol peptide (Navarro tum, the dorsal and lateral portions of the lateralet al., 2005) causes a significant rise in circulating LH septal nucleus contain moderate amounts of GnRHlevels. Almost all GnRH neurons express the receptor binding sites, whereas the medial septum and thefor kisspeptin, GPR54 (Messager et al., 2005; Navarro diagonal band are not labeled. In the hypothalamus,et al., 2005), and administration of kisspeptin causes a only the arcuate nucleus is labeled and, further cau-rapid and transient expression of the transcription dally, the interpeduncular nucleus and the central grayfactor fos (Irwig et al., 2004). This finding is remark- contain measurable amounts of GnRH binding because fos expression in GnRH neurons is lim- Specific binding is also seen in the cortical nucleus ofited to a few hours during the LH surge (Lee et al., the amygdala and more prominently in the hippocam-1990, 1992; Hoffman et al., 1993), and up to now only a pus, where most label is associated with the strata oriensstrict regimen of sequential estradiol and progester- and radiatum. The highest number of GnRH bindingone administration to ovariectomized rodents was sites is measured in the parasubiculum, which is labeledable to induce fos expression in GnRH neurons mim- at the medial tip of the caudal cerebral cortex ( Jennesicking the events that occur during the preovulatory et al., 1988).surge. Together, these data suggest that kisspeptin is apotent stimulator of GnRH release and that kisspep-tin plays an important role in the regulation of GnRH 51.3.2 Characterization of GnRHneurons under physiological conditions. However, Receptors in the Brainjust like the other neuropeptides or amino acid trans- Biochemical analyses of the specificity and affinity ofmitters, kisspeptin is not absolutely required since the GnRH binding to hippocampal membrane pre-GPR54-deficient mice, although infertile, respond parations show that they are very similar when com-to estradiol followed by progesterone administration pared to the anterior pituitary GnRH receptors inwith elevated LH levels and fos expression in the that all agonists that bind to hippocampal membranesGnRH neurons (Dungan et al., 2007). also bind to the pituitary GnRH receptors with
  • 11. The Gonadotropin-Releasing Hormone and Its Receptor 1655similar inhibiting concentration (IC50). In addition, dentate gyrus of the hippocampus in both male andGnRH fragments or different forms of GnRH from female rats ( Jennes et al., 1995). These data are inlower vertebrates do not bind to the hippocampal good agreement with studies that show a transientGnRH binding sites, nor do they interact with the elevation in the number of GnRH-binding sites inpituitary GnRH receptor. The only GnRH analog the hippocampus of castrated male rats (Badr et al.,that we found to bind to the pituitary GnRH receptor 1988; Ban et al., 1990). In the mediobasal hypothala-but not to hippocampal preparations is the antagonist mus, GnRH receptor mRNA levels were elevated in[D-p-Glul, D-Phe2, D-Trp3,6]GnRH ( Jennes et al., the morning of the estrogen- and progesterone-1990). The reason for this discrepancy is not known, induced LH surge, whereas the levels declined dur-but it is possible that the difference in the lipid ing the surge ( Jennes et al., 1997), which indicatesmicroenvironment may prevent the binding of this that the cells increase the synthesis of GnRH recep-analog to hippocampal binding sites. That the brain tor mRNA and probably protein in preparation forGnRH binding sites are very similar to the GnRH the LH surge and not as a consequence of the surge.receptor in the anterior pituitary was shown by in situ These data are similar to the results of Seong et al.hybridization that applied cRNA probes encoding (1998), who used competitive PCR to measurethe pituitary GnRH receptor. The results of these GnRH receptor mRNA in tissue punches of thestudies show that the mRNA is present in most areas mediobasal hypothalamus. These studies show thatof the brain where GnRH binding sites were mea- the administration of progesterone after estradiolsured, except for the interpeduncular nucleus and priming causes a dose-dependent decrease in GnRHthe central gray where no hybridization signal was receptor mRNA levels, whereas estradiol alone sti-detected. However, a hybridization signal was de- mulated GnRH receptor expression in the medioba-tected in the ventromedial nucleus and the medial sal hypothalamus. In addition, luciferase activityhabenula, and it was hypothesized that the GnRH under the control of the GnRH receptor promotorreceptor protein is synthesized in the neurons of the in transgenic mice is greatly enhanced in whole brainventromedial nucleus and transported anterogradely of ovariectomized animals that received estradiolto the central gray along a well-characterized exten- when compared to untreated ovariectomized micesive projection (Conrad and Pfaff, 1976). Similarly, (Duval et al., 2000). However, the data from thesethe protein could be synthesized in the neurons of studies with brain tissues are different from the datathe medial habenula and transported anterogradely obtained with pituitary tissue in which GnRH recep-through the fasciculus retroflexus to the interpedun- tor mRNA levels are low during the morning of thecular nucleus ( Jennes and Woolums, 1994). Addi- LH surge before they rise at noon and remain hightional studies are needed to determine if these until after the surge (Bauer-Dantoin et al., 1993). Thehypotheses are correct. The anatomical data describ- reasons for this discrepancy are not clear; it is plausi-ing the presence of GnRH receptor mRNA or protein ble that newly synthesized GnRH receptor protein inin the brain have been confirmed with polymerase the mediobasal hypothalamus is transported to distantchain reaction (PCR) for the mediobasal hypothala- sites, thus requiring more time before it reaches itsmus (Seong et al., 1998) and in transgenic mice that final location. That appropriate synthesis of thesynthesize luciferase under the control of the GnRH GnRH receptor in the brain may be important forreceptor promoter (Duval et al., 2000). Together, the occurrence of an estrogen- and progesterone-these studies clearly strengthen the view that specific induced LH surge is indicated by experiments thatreceptors for GnRH exist in select regions of the use the intraventricular administration of antisensecentral nervous system. oligonucleotides to reduce receptor synthesis. The administration of antisense oligonucleotides but not of sense oligonucleotides significantly decreases51.3.3 Regulation of GnRH Receptor GnRH receptor mRNA in the mediobasal hypothala-Expression in the Brain mus in estrogen- and progesterone-treated animalsVery little is known about the regulation of brain and causes a significant reduction in circulating LHGnRH receptor expression. We could show with (Seong et al., 1998). However, this treatment within situ hybridization that the removal of the gonads antisense oligonucleotides also reduced GnRHleads to a significant elevation of GnRH receptor receptor synthesis in the anterior pituitary; thus, it ismRNA levels in areas CAI and CA3 and in the not clear if the primary surge-blocking effect was
  • 12. 1656 The Gonadotropin-Releasing Hormone and Its Receptorexerted at the level of the central nervous system or activity of the target neurons. A change in the activitythe anterior pituitary. Together, these data suggest of the GnRH target neurons, many of which projectthat GnRH receptor mRNA and protein expression to the median eminence, could alter the release ofis at least in part regulated by gonadal steroids. Based GnRH, which controls pituitary gonadotrope func-on the anatomical distribution of estrogen receptors tion. Thus, GnRH neurons could participate in theand GnRH receptors, it is possible that the effects of regulation or synchronization of other GnRH neu-the gonadal steroids are exerted directly in the GnRH rons indirectly by using neurons in the arcuatereceptor synthesizing cells because ER-a is promi- nucleus as a relay station. However, many more stud-nently expressed in the arcuate nucleus, whereas ies are needed to establish the precise role thatER-b is the dominant form in the hippocampus GnRH plays inside the central nervous system.(Shughrue et al., 1997).51.3.4 Functional Aspects of GnRH 51.4 Molecular and CellularReceptors in the Brain Mechanism of GnRH Action in the Anterior PituitaryWe can only speculate about the function of GnRHreceptors in the brain. It has been known for many Much of the information regarding the molecularyears that peripheral or central administration of mechanism of GnRH has come from studies assessingGnRH can facilitate reproductive behaviors (Moss the coupling of the pituitary-derived receptor (Sealfonand McCann, 1973; Pfaff, 1973), especially when the et al., 1997; Byrne et al., 1999; Stojilkovick et al., 1994)peptide is administered into the central gray (for to effector systems. Pituitaries themselves have alsoreview, see Pfaff et al. (1994)). It is thought that the been a useful model, as have reconstituted systems incentral gray receives extensive afferent projections which receptors (including native, chimera, andfrom the ventromedial nucleus (Conrad and Pfaff, mutant receptor moieties) have been stably or tran-1976), which may transport the GnRH receptor siently transfected into systems that do not ordinarilyprotein. An enhanced GnRH receptor expression express receptors (Kaiser et al., 1997).in the neurons of the ventromedial nucleus duringthe morning of the LH surge could indicate thathigh levels of the receptor protein can reach the central 51.4.1 GnRH Receptorgray before estrus, which is the time of the cycle whenreproductive behaviors are most pronounced. The pituitary GnRH receptor has been cloned from The arcuate nucleus is one of the hypothalamic a substantial number of mammalian and premammalianregions that are essential for the maintenance species (Sealfon et al., 1997; Tsutsumi et al., 1992). Thisof regular estrous cyclicity (Bogdanove, 1964), and molecule is a member of the seven-transmembranethis nucleus contains a large number of different receptors superfamily. Because of the modest length ofneuropeptide- or monoamine-expressing neurons both the (extracellular) amino and (intracellular) car-(Chronwall, 1985), many of which also contain recep- boxyl terminal, the mammalian GnRH receptor type Itors for estradiol ( Jirikowski et al., 1986; Sar et al., (hereafter referred to only as GnRHR) is among the1990). Thus, the arcuate nucleus is generally thought smallest members of this superfamily (Ulloa-Aguirreof as a center in the brain in which the estrogen and Conn, 2000; Millar, 2003) and may approach thesignals are converted into neuronal signals. Among smallest size G-protein-coupled receptor (GPCR) thatthe estradiol target cells in the arcuate nucleus are can bind ligand and transduce an intracellular signal. Inneurons that contain NPY (Chronwall et al., 1985), birds, fish, and reptiles, the carboxyl tail is significantlyb-endorphin (Khatchaturian et al., 1985), or GABA longer, due to an extended carboxyl terminal, and this(Mugnaini and Oertel, 1985), all of which are known may reflect the altered regulation in these species,to affect GnRH release from the median eminence compared to mammals (Blomenrohr et al., 1999; Hed- ¨into the capillary plexus (for review, see Kordon et al. ing et al., 1998; Lin et al., 1998a; Millar, 2003). In the(1994)). If these neurons express GnRH receptors, as primate GnRHR, the expression levels are relativelyis suggested by the anatomical overlap of GnRH- low, an effect which appears attributable to the presencereceptor mRNA-containing neurons and the pepti- of an Lys191 not present in rat or mouse sequencesdergic and aminergic neurons, then GnRH released (Arora et al., 1999; Stanislaus et al., 1997). The removalin the arcuate nucleus could affect the secretory of this amino acid dramatically increases expression
  • 13. The Gonadotropin-Releasing Hormone and Its Receptor 1657levels, and there appears to be functional interac- et al., 1999; Thomas et al., 2007) and for heterogeneoustion between modifications at this and other sites receptor regulation (Rocheville et al., 2000a,b).(Stanislaus et al., 1997). 51.4.4 Receptor Trafficking51.4.2 Effector Coupling Endoplasmic reticulum qualityLike some other members of the GPCR’s superfamily, control system and the role of endogenousthe GnRHR appears functionally coupled to multiple chaperone proteinsG-proteins and appears significantly promiscuous, Molecular chaperones serve as a control mechanismdepending on the availability of G-proteins. Evidence for recognizing, retaining, and targeting misfolded pro-for coupling to multiple G-proteins comes from over- teins for their eventual degradation. These proteins areexpression and palmitoylation studies (Stanislaus key components of the endoplasmic reticulum (ER)et al., 1997, 1998), identification of separate sites of quality control system (QCS), a complex sorting sys-interaction (Ulloa-Aguirre et al., 1998; Arora et al., tem that identifies and separates newly synthesized1999), and other experimental approaches. This proteins according to their maturation status (Ulloa-observation has been an attractive consideration in Aguirre et al., 2004a). Although the steric character ofan explanation of how a single class of ligand inter- the protein backbone restricts the spectrum of proteinacting with a single class of receptor can regulate shapes that are recognized by the stringent qualitymultiple end points in coordinated, yet independent control mechanisms, some features displayed by pro-functions (release and biosynthesis of LH, FSH, and teins (including exposure of hydrophobic shapes,secretogranin; up- and downregulation of receptors; unpaired cysteines, immature glycans, and particularand sensitization and desensitization). Several reports sequence motifs) have been identified as important for(Kaiser et al., 1995; Pinter et al., 1999) in which differ- chaperone–protein association (Ellgaard and Helenius,ent techniques were used to provide variable levels of 2001; Dong et al., 2007). In fact, molecular chaperonesreceptor per cell suggest that differential regulation possess the ability to recognize misfolded proteins bymay be related to receptor number. In this way, the the exposure of hidden hydrophobic domains or spe-stimulus (GnRH) and the status of the target cell both cific sequences (Dong et al., 2007; Tan et al., 2004).participate in defining the responses elicited. Through this association, chaperones may stabilize unstable conformers of nascent polypeptides to pre- vent aggregation and facilitate correct folding, or51.4.3 Receptor–Receptor Interactions assembly, of the substrate via binding and release cyclesA series of observations dating back to the 1980s suggest (Hartl and Hayer-Hartl, 2002).the following: (1) Techniques that cause GnRHR to Several GPCR interacting proteins that supportassociate provoke all known actions of the releasing trafficking to the cell surface have been identified.hormone, even when the receptor is occupied by an Neither inactivation nor afterpotential A (Nina A), aantagonist (Conn et al., 1982b). (2) The occupancy of photoreceptor-specific integral membrane glycopro-the receptor by agonists is sufficient to cause receptors tein, is a molecular chaperone that facilitates cell- ˚to associate to within a distance of 50–100 A (Conn surface membrane expression of the sensory GPCRet al., 1982a,b; Cornea et al., 2001). (3) This process rhodopsin 1 in Drosophila melanogaster ; its absence(termed microaggregation, oligomerization, or, poten- leads to rhodopsin 1 ER accumulation and degrada-tially inaccurately, dimerization) is distinct from (and tion (Shieh et al., 1989; Schneuwly et al., 1989; Colleytemporally a precursor to) the process of patching, et al., 1991). Its mammalian homolog RanBP2 specif-capping, and internalization (macroaggregation) that is ically binds red/green opsin molecules and acts as aassociated with the extinction of the response system. chaperone aiding proper folding, transport, and local- These data are consonant with a role of microag- ization of the mature receptors to the cell membranegregation in GnRHR signaling. Evidence for receptor– (Ferreira et al., 1996). ODR4 is a molecular chaper-receptor interactions have now been observed for a one that assists in folding, ER exit, and/or targetingnumber of GPCRs (Conn et al., 1985; Pace et al., of olfactory GPCRs (e.g., ODR10 in the nematode1999; Harmatz et al., 1985; Hebert et al., 1996; Blakely Caenorhabditis elegans) to olfactory cilia (Dwyer et al.,et al., 2000; Heldin, 1995; Gether, 2000; Lee et al., 1998; Gimelbrant et al., 2001). Calnexin and calreti-2000b) and are suggested to be important for the expla- culin are molecular chaperones that bind a broadnation of independent mediation of responses (Pinter range of glycoproteins, including several GPCRs
  • 14. 1658 The Gonadotropin-Releasing Hormone and Its Receptor(e.g., the GnRHR, vasopressin-2 receptor, and the charge (12 instances); the remainder involved a gainglycoprotein hormones receptors) (Vassilakos et al., or loss of Cys (four instances) or a loss of a Pro (one1998; Schrag et al., 2003; Rozell et al., 1998; Morello instance). Each of these types of changes might beet al., 2001; Helenius et al., 1997; Brothers et al., expected to have a substantial impact on the overall2006). The action of these chaperones predominantly shape of the receptor, unlike a conservative substitu-centers on substrate N-glycans present on the newly tion (i.e., Gly for Ala) for example. As misshapenedsynthesized proteins, adding hydrophobicity to the (misfolded) proteins, the single amino acid mutantsfolding protein (Schrag et al., 2003; Rozell et al., 1998; characteristically failed the cellular QCS and mis-Morello et al., 2001; Helenius et al., 1997). When routing occurred (Ulloa-Aguirre et al., 2004a); in theN-linked glycosylation or early glycan processing cases examined, misrouting was due to retention infails, glycoproteins misfold, aggregate, and fail the the ER ( Janovick et al., 2003a; Leanos-Miranda et al., ˜QCS (Morello, et al., 2001). Receptor activity modify- 2003, 2005; Ulloa-Aguirre et al., 2004b).ing proteins (RAMPs) are proteins that interact with The corollary of the observation that these areseveral GPCRs (e.g., the calcitonin receptor-like misrouted proteins, is that, if restored to the plasmareceptor, the vasoactive intestinal polypeptide/pitui- membrane, these mutants can become functionaltary adenylate cyclase-activating peptide receptor, the ( Janovick et al., 2003a; Leanos-Miranda et al., 2003, ˜glucagon receptor, and the parathyroid hormone 2005). This happens because they have retained (orreceptor) fostering the transport of the associated regain) both the ability to bind agonist and the abilityreceptor to, and regulating its signaling function at, to transduce a signal. This differs from a prior viewthe PM (Christopoulos et al., 2003), whereas gC1q-R that most mutants are defective because they have(receptor for globular heads of C1q) interacts with the (permanently) lost the ability to bind ligand or cou-carboxyl terminus of the alpha1B-adrenergic receptor ple to effector proteins. The ability to rescue andand regulates the maturation and expression of the restore proteins to function opens a new avenue inreceptor (Xu et al., 1999). Another molecular chaper- therapeutics which appears broadly applicable toone is BiP/Grp 78, which is involved in the protective disease-causing mutants of the GnRHR.unfolded protein response; a cell stress program acti- Restoration of binding and coupling activity ofvated when misfolded proteins accumulate and/or mutants was initially shown by adding plasma mem-aggregate in the ER (Yang et al., 1998; Schroder and brane targeting sequences to defective humanKaufman, 2005). Finally, DriP78 is an ER-membrane- GnRHR mutants (i.e., making chimeras of humanassociated protein that binds to the F(x)3F(x)3F motif mutants with the C-terminal sequence found in cat-of the dopamine receptor (and presumably other fish GnRHR that increases plasma membrane target-GPCRs bearing this motif), thereby facilitating its ing of the GnRHR (Lin et al., 1998b)), or by deletingmaturation and export to the PM (Bermak et al., an amino acid (K191) found in the human (and other2001). Identification of these particular molecular primates) wild-type (WT) receptor that, when pres-chaperones is important since they represent a poten- ent, decreases routing to the plasma membrane. Suchtial target to manipulate ER retention and/or export modifications led to rescue of the mutant sequencesmechanisms, and hence a means for influencing pro- as assessed by both radioligand binding and by thetein trafficking and secretion (Aridor and Balch, 2000; ability of agonist to activate Gaq/11-protein-mediatedRivera et al., 2000). inositol phosphates (IP) production (Lin et al., 1998b; Janovick et al., 2003b). Mutant GnRHRs isolated from More recently ( Janovick et al., 2003a, 2007; Leanos- ˜patients with hypogonadotropic Miranda et al., 2003, 2005), we learned that it is possi-hypogonadism are actually misfolded and ble to use a pharmacological approach and show thatmisrouted proteins that can be rescued and small, nonpeptide molecules (pharmacologic chaper-restored to function ones or pharmacoperones) which bind the GnRHRResults over the last 7 years have led to the conclu- mutants can stabilize most of the mutant proteinssion that all of the first 17 reported single amino acid (15 of 17) in a conformation that passes the QCS;mutations of the human GnRHR that lead to hypo- these rescued mutants are not retained in the ER, butgonadotropic hypogonadism (HH) are actually mis- route correctly to the plasma membrane, where theyfolded proteins ( Janovick et al., 2002). All 17 genetic bind ligand (shown with radioligand) and successfullymutations resulted from single amino acid changes in transduce signaling (shown by IP response). Such phar-the GnRHR. Most frequently, this was a change in macoperones are, of course, lead drug candidates.
  • 15. The Gonadotropin-Releasing Hormone and Its Receptor 165951.4.4.3 The ability to rescue mutant Castro-Fernandez et al., 2005). The ability to rescueproteins using pharmacoperones has misfolded proteins with drugs presents a new thera-therapeutic potential peutic approach for a remarkably diverse palette ofPharmacoperones for the GnRHR mutants have now diseases caused by other misfolded molecules: Alz-been identified from three different chemical classes heimer’s disease, cataracts, cystic fibrosis, familial(indoles, quinolones, and erythromycin macrolides; hypercholesterolemia, HH, nephrogenic diabetesJanovick et al., 2003a) using combinational chemical insipidus, and retinitis pigmentosa, among otherslibraries. Within each chemical class, there is an (Ulloa-Aguirre et al., 2003, 2004a,b; Conn and Jano-effect of mutant rescue that is proportional to the vick, 2005; Maegawa et al., 2007; Lumb et al., 2003; Vijaffinity of binding to the receptor ( Janovick et al., et al., 2006; Biswas et al., 2004; Biswas and Das, 2004;2003a). In retrospect, it is not surprising that concen- Kim et al., 2006; Lashuel and Hirling, 2006; Cashmantrations needed for rescue differ between chemical and Caughey, 2004; Mallucci and Collinge, 2005). Inclasses of pharmacoperones since it is likely that the the case of the human GnRHR, this receptor alsospecific chemical interaction between the drugs and appears to provide opportunities for regulation bythe mutants would stabilize the mutants by distinct intentionally misfolding a portion of the WT receptor,means (i.e., by interacting with different residues on resulting in a circumstance in which a proportionthe receptor). Only two of the mutants in the group of the synthesized molecule is retained in the ERdiscussed (the first 17 GnRHR mutants reported, ( Janovick et al., 2006; Conn et al., 2006a,b). Otherexcluding deletion and truncation mutants) cannot GPCRs ( Janovick et al., 2006) appear to share thebe at least partially rescued by this (pharmacoperone) balance between the ER and the plasma membraneapproach ( Janovick et al., 2006). In these two cases, and therefore these wild-type receptors may be candi-this occurs because the mutation leads to a change dates for this rescue approach – when the therapeuticthat is so distorting to the protein structure that rescue goal is to increase the plasma membrane expression ofdoes not occur. In both of these cases (amino acids 168 these moieties.and 217), the mutation is identical, Ser ! Arg and both Accordingly, it is useful to consider potential usesmutations occur in transmembrane segments (trans- (Conn et al., 2007) of pharmacoperones in four groups:membrane segments 4 and 5, respectively), resulting (1) to prevent misfolding of molecules that do soin a highly unfavorable thermodynamic change that and lead to disease (e.g., cataracts and neurodegenera-causes a specific ( Janovick et al., 2006) effect on the tive diseases); (2) to rescue mutants (e.g., mutantreceptor configuration (Knollman et al., 2005). GnRHR in HH, cystic fibrosis transmembrane con- Because the pharmacoperones are successful in res- ductance regulator in cystic fibrosis, a-galactosidase Acuing the remaining 15 mutants – even though the loci in Fabry’s disease); and (3) to increase (or decrease) theof the mutations are widely distributed over the recep- percentage of WT molecules that route to the mem-tor – this argues for the generality of this approach for brane, when only a fraction of the total synthesizedmutants of the human GnRHR. Moreover, the drugs material is expressed at the plasma membrane, as isused as pharmacoperones were initially developed as the case for the WT hGnRHR (Conn et al. 2006a,b).oral antagonists of GnRH for use in humans; accord- There is a fourth condition in which perturbation ofingly, there is extensive safety and pharmacokinetic and the ER homeostasis results in disorders because of thepharmacodynamic data available in human and animal promotion in the synthesis of deleterious productsmodels which will make it easier to move the rescue potentially treatable with pharmacoperones; a goodtechnique to in vivo rescue situations. example of this is prion replication (Hetz et al., 2007). The rescue approach appearsgenerally applicable to other mutant GPCRs, Acknowledgmentsnon-GPCR receptors, ion channels, andenzymes associated with disease: This research was supported by NIH AG025647,This supports the importance of AG17164 and RR15592(L.J.) NIH HD-19899, TW /understanding the mechanism of this event HD-00668, RR-00163, and HD-18185 (P. Michaelin a well-defined system Conn), and grant 45991 from CONACyT, MexicoMutants of other GPCRs, non-GPCR receptors, as (Alfredo Ulloa-Aguirre). Alfredo Ulloa-Aguirre is awell as ion channels and enzymes can be rescued recipient of a Research Career Development Awardby this approach and restored to activity (e.g., ´ ´ from the Fundacion IMSS, Mexico.
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  • 24. 1668 The Gonadotropin-Releasing Hormone and Its ReceptorXu Z, Hirasawa A, Shinoura H, and Tsujimoto G (1999) hormone-immunoreactive neurons in the medial olfactory Interaction of the alpha(1B)-adrenergic receptor with gClq-R, placode and basal forebrain of embryonic mice. a multifunctional protein. Journal of Biological Chemistry Neuroscience 46(2): 407–418. 274: 21149–21154.Xu M, Urban JH, Hill JW, and Levine JE (2000) Regulation of hypothalamic neuropeptide Y Yl receptor gene expression during the estrous cycle: Role of progesterone receptors. Further Reading Endocrinology (Baltimore) 141(9): 3319–3327.Yang Y, Turner RS, and Gaut JR (1998) The chaperone BiP/ Ulloa-Aguirre A, Janovick JA, Miranda AL, Conn PM (2006) GRP78 binds to amyloid precursor protein and decreases G protein-coupled receptor trafficking: Understanding the Ab40 and Ab42 secretion. Journal of Biological Chemistry chemical basis of health and disease. ACS Chemical Biology 273: 25552–25555.Zheng LM, Pfaff DW, and Schwanzel-Fukuda M (1992) Electron 1(10): 631–638. microscopic identification of luteinizing hormone-releastng
  • 25. Biographical SketchLothar Jennes is professor in the Department of Anatomy and Neurobiology at the University of Kentucky. He received hisgraduate training at the University of Salzburg, Austria, and postdoctoral training at INSERM in Lille, France, theUniversity of North Carolina in Chapel Hill, and Duke University in Durham. His laboratory focuses on theneuroendocrine control of reproduction with particular emphasis on the factors and mechanisms that controlgonadotropin-releasing hormone neurons in young and senescent animals. He is the author or co-author of over 100research publications.Alfredo Ulloa-Aguirre was born in 1949 in Mexico City. He obtained his MD, DSc, and specialty degrees in Internal Medicineand Reproductive Endocrinology at the National University of Mexico. In 1980 he was awarded a Rockefeller FoundationFellowship to attend the Division of Reproductive Biology of the University of Pennsylvania in Philadelphia, Pennsylvania,USA, as a posdoctoral fellow in reproductive biology and endocrinology. In 1996 and 2003, he was a visiting professor at theOregon National Primate Research Center, Oregon Health Sciences University in Beaverton, Oregon, USA where he iscurrently a collaborating scientist. He leads the Research Unit in Reproductive Medicine at the Mexican Institute of SocialSecurity and has published prolifically and received many awards and honors for his scientific achievements. He has been amember of many editorial boards including Archives of Medical Research, Human Reproduction, Endocrine, Reproductive BiomedicineOnline and Reproductive Biology and Endocrinology, and Recent Patents on Endocrine, Metabolic & Immune Drug Discovery. The researchfocus of his group includes the study of the structure–function relationship of gonadotropins and gonadotropin receptors, theGnRH receptor, and the neuroendocrine regulation of gonadotropin secretion.Jo Ann Janovick is a senior research associate and registered pharmacist and has been in the Conn laboratory for 23 years,contributing to research, technique development, and training. She is a collector of Pacific Northwest Indian art and isdevoted to her two dogs.Valeriya V. Adjan, MD, is a scientist II in the Department of Anatomy and Neurobiology at the University of Kentucky.Before joining the University of Kentucky in 2002, she was an internal medicine physician in Rostov, Russia. At UK, shebecame interested in the field of reproductive biology and joined the Jennes lab to study the role of neurotransmitters thatcontrol GnRH release.P. Michael Conn is associate director and senior scientist of the Oregon National Primate Research Center and professor ofphysiology and pharmacology, and cell and developmental biology at OHSU. He has written extensively on the molecularmechanism of hormone action and on topics related to neuroscience, endocrinology, physiology, pharmacology, andmolecular biology. He is an outspoken supporter of the humane use of animals in research and the importance of publiceducation about the importance of science.