Structural and Functional Evolution of Gonadotropin-Releasing Hormone

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. I W

Structural and Functional Evolution of Gonadotropin-Releasing Hormone ROBERTP. MILLARA N D JUDY A. KING Medical Reseurch Council Regulatory Peptides Research Unit, Department of’ Chemical Pathology, University of Cape Town Medical School and Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa

I. Introduction Gonadotropin-releasing hormone (GnRH)I is a hypothalamic decapeptide (Fig. I ) which regulates reproduction by stimulating the release of pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which in turn stimulate gonadal activity (Schally, 1978). Following the structural elucidation of GnRH from porcine and ovine hypothalamus, it became generally accepted that the decapeptide was a unique molecular form. This belief arose from the following: 1. Studies purported to have shown nonribosomal biosynthesis of GnRH and thereby excluded the possibility of ribosomally biosynthesized prohormonal forms. 2. It was assumed that GnRH was confined to the central nervous system and, thus, was unlikely to be present in other tissues in a modified form. 3. Immunological and low-resolution chromatographic studies demonstrated that GnRH in the hypothalamus of nonmammalian vertebrates was identical to the mammalian peptide. This notion of a lack of interspecific differences in GnRH structure in vertebrates was supported by the demonstration that mammalian GnRH was biologically active in a wide range of mammalian species and in nonmammalian vertebrates (Schally, 1978).

A number of factors argued against the view of a nonribosomal synthesis of GnRH and a universal conservation of the GnRH structure.

( I ) The GnRH structure, comprising exclusively L-amino acids, a-amino peptide linkages, and a cyclized NH,-terminal Glu and COOH-terminal amide, is characteristic of ribosomally synthesized peptides. In contrast, ‘Abbreviations: GnRH, Gonadotropin-releasing hormone; mGnRH, mammalian GnRH: cGnRH 1. chicken GnRH I (Gln‘--GnRH); cGnRH 11. chicken GnRH I I (His’, Trp’. TyPGnRH): sGnRH. salmon GnRH (Trp’, Leun-GnRH); LH. luteinizing hormone; FSH, folliclestimulating hormone; HPLC. high-performance liquid chromatography. I49

Copyright Q 1987 by Academic Press. Inc. All riphls of reproduction in any form reserved.

ROBERT P. MILLAR A N D JUDY A. KING

I50 Pig/Sheep

pGlu'

-

His2

-

T r p 3 - Serb - T y r 5

-

Gly6 - Leu7 - Arg8 - Pro9 - Glyl0.NH2

Chicken I

Gln

Chicken I 1

His

Trp Trp

Salmon FIG.

I.

- Tyr - Leu

Structures of vertebrate GnRHs.

the presence of D-amino acids (e.g., in prokaryote antibiotic peptides) and unusual peptide linkages (y-glutamyl in glutathione and p-alanyl in carnosine in eukaryotes) characterizes peptides synthesized by nonribosomal mechanisms. (2) The related neurohypophyseal peptides, vasopressin and oxytocin, had been shown convincingly to be processed by proteolytic cleavages from ribosomally synthesized precursors. Moreover, the neurohypophyseal peptides exhibit structural heterogeneity in vertebrates, which is consistent with conservative single nucleotide base changes in the triplet codons (Acher, 1983). (3) Accepting that GnRH is synthesized ribosomally, it appeared likely that different GnRH molecular forms would have arisen in vertebrates during 400 million years of evolution, as is the case with the neurohypophyseal hormones (Acher, 1983). Research over the past 7 years has now established that there is considerable diversity in the structure of GnRH and related molecular forms. Higher molecular-weight prohormonal forms have been demonstrated, while structural variations in vertebrate hypothalamic GnRHs have been shown in several species from the major vertebrate classes. These structural differences have been confirmed by the isolation and structural analysis of GnRHs from a single species of bird, amphibian, and teleost (Fig. I). GnRH-like peptides, which may differ structurally from the mammalian hypothalamic peptide, have also been found in mammalian tissues, such as the extrahypothalamic brain, testis, and ovary. In this report, we review current knowledge on the molecular heterogeneity of GnRH, the biological actions of GnRH and related molecules, structure-activity relations, and the evolution of the hormone. 11. Structure and Distribution of GnRH and Related Molecular Forms

A. PROHORMONAL GnRH The existence of higher molecular-weight immunoreactive forms of GnRH in hypothalamic extracts suggested that these might constitute prohormonal species. In both rat and sheep hypothalami, immunoreactive

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

I5 I

GnRH peptides of molecular weights of >5K, 3K, and 2K were detected in addition to decapeptide GnRH (Millar et al., 1977, 1978, 1981a). Since the 5K peptide eluted in the void volume of a Sephadex G-25 column, it was possible that it had a molecular weight of greater than 5K. Recent studies in our laboratory using Sephadex G-50 and high-performance liquid chromatography (HPLC) gel permeation chromatography under denaturing conditions indicate that the precursor is approximately 8K-1OK in size. Extensive studies provided evidence for the prohormonal nature of the largest peptide. The peptide was specifically retained on immunoaffinity columns using an appropriate antiserum directed toward the central sequence of GnRH; it was not dissociated by rigorous treatment with 8 M urea and 6 M guanidinium hydrochloride and was not an immunoassay artifact, as it did not bind or degrade '251-labeledGnRH. Physiological manipulations, which altered hypothalamic GnRH content, also changed the occurrence of the putative prohormonal forms, and in tissues lacking GnRH, no immunoreactive higher molecular-weight material was detected (Millar et al., 1978, 1981a). Specific chemical modifications of each of the amino acids comprising the GnRH sequence resulted in a similar loss of immunoreactivity of both GnRH and the 25K prohormonal form when quantitating with appropriate antisera requiring the particular residues for binding (Millar et al., 1978, 1981a). These studies also demonstrated that proteolytic cleavage of GnRH and the prohormones with a range of enzymes (except trypsin) yielded appropriate losses of immunoreactivity with the different antisera. The presence of relatively higher proportions of the prohormonal forms in hypothalamic regions containing GnRH cell bodies compared with a paucity of prohormonal forms in the stalk median eminence, supported the classical concept of neuronal precursor-peptide processing for GnRH biosynthesis (Millar et al., 1978, 1981a). These observations have been more thoroughly demonstrated by immunocytochemistry (King and Anthony, 1983, 1984). The prohormonal forms were also more prevalent in the microsomal fraction than in purified synaptosomes (nerve endings) which contained exclusively fully processed decapeptide GnRH (Millar et al., 1981a). HPLC separation of the synaptosomal extract confirmed that only decapeptide GnRH is present in contrast to the presence of both somatostatin-28 and somatostatin-14 in synaptosomes (Kewley er al., 1981). Other studies addressed the question as to the localization of the GnRH sequence within the prohormonal peptide. The interaction of the 35K putative prohormonal GnRH species with seven region- and/or conformation-specific GnRH antisera indicated that the molecule is modified at both the NH2- and COOH-termini (Millar et al., 1978, 1981a). This indication of both NH2- and COOH-terminal extensions to the GnRH sequence was supported by the demonstration that aminopeptidase and carboxy-

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ROBERT P. MILLAK A N D JUDY A. KING

peptidase digestions led to increases in immunoreactivity (Millar er a/., 1978, 1981a). The 5K GnRH species was partially converted by hypothalamic peptidases into an immunoreactive peptide eluting in the position of GnRH. Progressive trypsin digestion released a 2K- to 3K- immunoreactive species followed by complete conversion to a single form migrating in the position of GnRH on Sephadex (3-25 (Millar et al., 1977, 1978, 1981a). It was suggested, therefore, that there are both NH,- and COOHterminal modifications or extensions to GnRH in the putative prohormonal GnRH and that trypsin-sensitive cleavage sites (basic amino acids) are interposed between the GnRH sequence and peptide extensions. This arrangement is common to many prohormones which are processed by trypsin-like cleavages at pairs of basic amino acids, followed rapidly by removal of the exposed basic residues by carboxypeptidase-B-like activity (reviewed by Douglass et al., 1984). It was also proposed that prohormonal GnRH is characterized by Gln in position one of the GnRH sequence, which is spontaneously (or enzymatically) cyclized after cleavage of the NH,-terminal extension to give rise to pGlu' (Millar er a/., 1981a). In addition, the amide (GIY'~*NH,) was presumed to arise from the amino group of an additional Gly preceding the basic residues. This now seems a likely possibility, as structural analysis of prohormonal forms of 1 I propeptides with COOH-terminal amides has revealed that all have this additional Gly, and recent studies on a pituitary amidating enzyme using synthetic peptide substrates have demonstrated an absolute requirement for Gly in this oxidative transamidation (Bradbury et d . , 1982). Subsequent studies demonstrated 26K and I .8K higher molecular-weight immunoreactive forms of GnRH in extracts of rat hypothalamus, cortex, and placenta (Gautron et al., 1981). The 26K peptide was recognized almost exclusively by NH,-terminus-directed antiserum, suggesting it is COOH-terminally extended. Unfortunately, the authenticity of the 26K species is uncertain, as rigorous dissociating conditions were not used. It is also uncertain as to whether both NH,- and COOH-extensions were present, as a middle-directed antiserum which would recognize such forms was not employed. The primary immunoprecipitable GnRH translation product of mouse and human hypothalamic mRNA in reticulocyte lysate was recently shown to be a 28K peptide (Curtis and Fink, 1983; Curtis et a / . , 1983). Allowing for the cleavage of a signalAeader sequence, this is comparable to the 26K peptide reported above. Although the above studies provided a persuasive argument for the existence of prohormonal GnRH, final proof lies in the isolation and sequence analysis of the putative prohormone and demonstration of its conversion to GnRH by the tissues concerned. The demonstration of incorporation of [3H]tyrosine into GnRH by human placental trophoblast (Tan and

I53

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

Rousseau, 1982) indicates the potential of this tissue for these biosynthetic and processing studies. An alternative approach to establishing the structure of prohormonal GnRH is by recombinant DNA technology. Several laboratories initiated programs aimed at the difficult goal of elucidating the sequence of GnRH mRNA. Our laboratory was unable to convincingly demonstrate GnRH clones in hypothalamic cDNA libraries using labeled synthetic oligomers (17-mers) coding for both NH,- and COOH-terminal sequences of GnRH. However, several clones which hybridized with these oligomers were identified in a cDNA library of a human buccal tumor cell line which produced GnRH. Recently, a major breakthrough occurred in establishing the sequence of a human GnRH gene and human placental mRNA encoding a precursor form of GnRH (Seeburg and Adelman, 1984). The cDNA sequence codes for a protein of 92 amino acids in which the GnRH decapeptide is preceded by a signal peptide of 23 amino acids and followed by a Gly-Lys-Arg sequence, as expected for enzymatic cleavage of the decapeptide from its precursor (Gubler et al., 1983) and arnidation (Bradbury et ul . , 1982) of the COOH-terminus of GnRH (Fig. 2). The next apparent cleavage site is Ly~'"-Lys''~,which suggests that a 53 amino acid peptide is also processed from the precursor. Single basic amino acid residues (Fig. 2) might, however, also be cleavage sites as in several other prohormones. Since hormone precursors can generate several biologically active peptides, we recently synthesized the sequence 14-26 and tested the effects of this peptide on cultured human pituitary cells. The peptide did not affect thyrotropin or prolactin release but, surprisingly, stimulated both LH and

PRE -23

GnRH -1 1

CS 10

11

Gh H s TP Ser Tyr oly Leu Arg Pro oly Gly lys Arg

14

CARBOXYL-TERMINAL EXTENSION 27 37 46 50 53 66 LYS

Arg

Arg ArgLys

PCS 69

lys lys le

FIG.2. Schematic diagram of the structure of the human placental GnRH precursor as determined by nucleic acid sequencing of the corresponding cDNA (Seeburg and Adelman, 1984). The precursor consists of a signal sequence (PRE) of 23 amino acids followed immediately by the GnRH decapeptide sequence. Cleavage of the signal peptide reveals a NHZ-terminusGin which cyclizes (enzymatically or spontaneously) to pyro-Glu. The GnRH sequence is followed by a Gly. which is the donor for the COOH-terminal amide of GnRH, and Lys-Arg, which is a conventional dibasic amino acid cleavage site (CS). This is followed by a 53 amino acid peptide before a second potential cleavage site, suggesting that this peptide is released together with GnRH. The positions of single basic amino acid residues are also shown, as these may be cleavage sites as in the precursors of vasopressin, enkephalins, somatostatin. cholecystokinin, growth hormone-releasing factor, epidermal growth factor, and nerve growth factor.

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ROBERT P. MILLAR A N D JUDY

A.

KING

FSH release (R. P. Millar, P. J. Wormald, and R. C. deL. Milton, unpublished). GnRH antagonists did not affect the stimulation, suggesting that the effects are mediated by a separate receptor. The physiological significance of the presence of a second gonadotropin-releasing peptide in the GnRH precursor requires clarification.

B. GnRH IN MAMMALIAN TISSUES 1. Hypothalamic GnRH GnRH was originally isolated from porcine hypothalami by virtue of its ability to stimulate the release of pituitary gonadotropic hormones (Matsuo et al., 1971; Scha4ly et al., 1971). Subsequently, the same structure was established for ovine hypothalamic GnRH (Amoss et al., 1971). Hypothalamic GnRH in several other mammalian species has identical chromatographic and immunological properties to porcine-ovine GnRH (designated mGnRH) (Fig. 1). 2 . Extrahypothalamic Brain GnRH The presence of immunoreactive GnRH in extrahypothalamic brain regions is well documented (Endroczi and Hilliard, 1965; Silverman and Krey, 1976; Ibata et al., 1983), but few studies have investigated the molecular nature of the peptide(s). The sheep pineal gland has a GnRH molecular form which is identical to the hypothalamic peptide in its interaction with different region-specific antisera and which cannot be distinguished using gel filtration chromatography, cation-exchange chromatography, or higher resolution HPLC (King and Millar, 1981a; Millar and Tobler, 1981; Millar et al., 1981b). These studies demonstrated a second form of GnRH which was structurally distinct from the hypothalamic hormone. This GnRH species was of similar size to GnRH as it comigrated with the decapeptide on Sephadex G-25, brlt was less positively charged and eluted earlier on cation-exchange chromatography. The peptide had similar properties to a chicken hypothalamic GnRH (Gln8-GnRH) (see below) in coeluting on cation-exchange chromatography and reversed-phase HPLC. Interactions with NH2- and COOH-terminal-directed antisera and resistance to degradation by aminopeptidase and carboxypeptidase A supported the studies with antisera, which indicated the presence of pGlu' and Gly" NH, in the molecule. The pineal gland form of GnRH had intrinsic LH-releasing activity, but decreased the LH response to GnRH, suggesting that it may be a weak agonist. The presence of Gln8-GnRH in the sheep pineal gland has recently been confirmed using high-resolution HPLC systems, which were specifically designed to separate GnRH an-

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

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alogs (J. A. King and R. P. Millar, unpublished). The occurrence of a GnRH species in the pineal gland, which is similar to hypothalamic GnRH of a lower vertebrate (Gln'-GnRH in chicken hypothalamus), is reminiscent of reports on the presence of vasotocin in the mammalian pineal gland (Pavel, 1979). The finding of Gin'-GnRH in the pineal gland poses the possibility that this molecular species is present in other areas of the nervous system and serves a neurotransmitter or neuromodulator role. 3. Gonadal GnRH

GnRH has direct effects on the gonads of laboratory animals (Hsueh and Erickson, 1979; Clayton et al., 1981; Sharpe and Cooper, 1982), and high-affinity binding sites for GnRH analogs have been demonstrated in the Leydig cells of the testis (Labrie et al., 1978; Bourne et al., 1980; Perrin et al., 1980; Sharpe and Fraser, 1980; Fraser et al., 1982; Millar ef al., 1982) and in the granulosa cells of the ovary (Harwood et al., 1980; Pieper el al., 1981; Hazum and Nimrod, 1982; Marian and Conn, 1983). Testicular extracts were reported to displace '2'I-labeled GnRH in radioreceptor assays (Sharpe et al., 1981). Immunoreactive GnRH species from acetic acid-extracted and immunoaffinity-purified rat testicular material (Dutlow and Millar, 1981) have been characterized by region-specific antisera and by gel filtration and HPLC. Molecular species of -IOOK, 32K, 5K, and IK were all found to interact strongly with a COOH-terminaldirected antiserum and poorly with middle- and NH,-terminal-directed antisera, suggesting they contain the COOH-terminal sequence of hypothalamic GnRH. Only the lower molecular-weight forms displaced "'Ilabeled GnRH binding to rat pituitary GnRH receptors. A recent report of a similar study on rat testis extracts also suggests that the testicular material shares COOH-terminal sequences in common with GnRH and displaces "'I-labeled GnRH in the radioreceptor assay (Bhasin el al., 1983). In contrast, another study on rat testicular GnRH showed that the high-molecular-weight form could be dissociated to molecules of the same size as GnRH, which reacted more poorly with COOHterminal-directed antisera than with an antiserum specific for the NH2and COOH-terminus and one recognizing the middle region of the molecule (Paul1 et al., 1981 ;Turkelson et al., 1983). In porcine follicular fluid, three immunoreactive GnRH peptides in the molecular range 30K-50K have been detected. The immunoreactivity of these peptides was found to increase after trypsin digestion (M. S. Hendricks and R. P. Millar, unpublished). Since our recent studies with hypothalamic prohormonal GnRH have shown that rigorous conditions are necessary to dissociate high-molecular-weight forms of GnRH, much of the work on gonadal GnRH should be regarded with caution. This is emphasized by the studies of Turkelson

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ROBERT P. MILLAR AND JUDY A. KING

et al. (1983). Since the gonadal GnRH examined may be bound to other peptides, the antisera studies defining sequences in common with hypothalamic GnRH must also, therefore, be regarded with caution, as certain sequences may be obscured by the binding peptide.

4. GnRH in Other Tissues Immunoreactive GnRH detected in the placenta has immunological, chromatographic, and biological properties identical to the hypothalamic decapeptide (DePalatis et al., 1980; Khodr and Siler-Khodr, 1980; Lee el al., 1981;Tan and Rousseau, 1982). Higher molecular-weight forms (26K) have also been detected in this tissue (Gautron et al., 1981). Elucidation of the sequence of human placental GnRH cDNA (Seeburg and Adelman, 1984) has now confirmed the existence of a GnRH in the placenta with the identical structure of the hypothalamic peptide. The cDNA sequence codes for a 69 amino acid precursor (-8K). GnRH-like immunoreactive material has been demonstrated by radioimmunoassay or immunohistochemical techniques in the mammalian pancreas (Seppala and Wahlstrom, 1980a), submandibular gland (C. Dutlow and R. P. Millar, unpublished), olfactory system (Phillips et al., 1980; Dluzen and Ramirez, 1983; Witkin and Silverman, 1983), milk (Baram et al., 1977; Sarda and Nair, 1981; Amarant et al., 1982; Hazum, 1983), and in certain mammary tumors (Seppala and Wahlstrom, 1980b). Specific binding sites for GnRH have been demonstrated in the placenta (Currie et al., 1981; Belisle et al., 1984) and adrenal gland (Bernard0 et a / . , 1978; Pieper et al., 1981; Eidne et al., 1985a). A GnRH-like peptide has been described in the amphibian adrenal gland (see below), but there are no reports of GnRH in the mammalian adrenal gland. C. INTERSPECIFIC HETEROGENEITY IN GnRH

I . Birds In the early studies on bird hypothalamic GnRH, one study suggested an identity (Jeffcoate et al., 1974) of bird GnRH with mammalian GnRH (mGnRH), but others indicated that there were structural differences (Jackson, 1971; King and Millar, 1979a, 1980; Hattori et al., 1980). Chicken (Gallus domesticus) and pigeon (Colurnba livia) hypothalamic GnRHs had a similar molecular size to the mammalian peptide, but were less positively charged and differed immunologically (King and Millar, 1979a, 1980). Antisera directed toward the middle region of mGnRH and those recognizing the COOH-terminal three amino acids gave lower quantitation and nonparallel radioimmunoassay displacement curves. Antisera requiring the

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

I57

extreme NH,- and COOH-termini and tolerant of certain amino acid substitutions in the middle region of the molecule gave high quantitation and parallel displacement curves (King and Millar, 1979a, 1980). Examination of overlapping sequence requirements of these antisera indicated that the alteration in this form of chicken hypothalamic GnRH, designated chicken GnRH I (cGnRH I), resided in the position of Arg' (Fig. I ) . The difference was investigated in more detail by noting the interaction with the different antisera after selectively modifying the molecule at constituent amino acid residues of GnRH in turn by specific chemical and enzymatic treatment (King and Millar, 1982a). These data showed that cGnRH I differed at Arg'. The difference in isoelectric points of cGnRH I and mGnRH was compatible with a neutral amino acid substitution for Arg' of mGnRH. On the basis of evolutionary probability of amino acid interchange for Arg, Gln was a likely candidate. The putative cGnRH I (Gld-GnRH) (Fig. I ) was synthesized and shown to have identical immunological, chromatographic, and biological properties to natural cGnRH I (King and Millar, 1982a). Other GnRH analogs with substitutes of Ser, Trp, Leu, Met, Ile, Phe, His, Asn, Glu, Cit, Orn, and Lys in position eight had properties different from that of natural cGnRH I. The use of a specific antiserum raised against synthetic Gln'-GnRH, which has an absolute requirement for glutamine in position eight (King et al., 1983), confirmed the assignment of glutamine to position eight. Concurrent studies culminated in the purification of 17 pg of cGnRH I from 250,000 chicken hypothalami using a combination of affinity chromatography, cation-exchange HPLC, and reversed-phase HPLC. Amino acid analysis of an acid hydrolysate showed an absence of Arg and the presence of an additional Glu, compatible with the proposed structure (King and Millar, 1982b,c). Sequence analysis was consistent with the location of Gln as a replacement for Arg in the eighth position (King and Millar, 1982b; J. Spiess, J. A. King, and R. P. Millar, unpublished). Another group has confirmed the structure of this cGnRH I as Gln8-GnRH (Miyamoto et al., 1983). Subsequently, a second form of GnRH, His',Trp',Tyr'-GnRH [designated chicken GnRH I1 (cGnRH 11) (Fig. I I], was isolated from chicken hypothalamus (Miyamoto et al., 1984). Both forms of GnRH stimulate gonadotropin secretion, but it remains to be determined whether only one or both forms are released into the hypothalamopituitary portal system and reach the pituitary gland. GnRH has not been isolated from any other species of birds. Recent studies using chromatographic, immunological, and bioassay assessment have established the presence of cGnRH I and cGnRH I1 in ostrich (Struthio cumelus) hypothalamus (R. C. Powell, R. P. Millar, and J. A. King, unpublished) (Table I).

ROBERT P. MILLAR A N D JUDY A. KING

I58 hYLOGENETIC

TABLE I DISTRIBUTION OF THE FOURKNOWN VERTEBRATE BRAINGnRHs GnRH structure

Group Mammal Bird Reptile

Amphibian Teleost

Elasmobranch

Species

.

Pig" Sheep" Chicken" Ostrich (Strurhio camelus)b Alligator (Alligator mississippiensis)b Lizard (Podarcis sicula sicuIa)b Lizard (Cordylis nigra)b Skink (Calcides ocellatus)b Frog (Rana cafesbeiuna)" Toad (Xenopus l a e W b Salmon (Oncorhynchus ketay Codfish (Gadus morhua morhua)" Hake (Merluccius capensis)b Tilapia (Tilapia sparrmanii)b Coris julis" Mullet (Mugil cepha/us)b Milkfish (Chanos chanos)b Trout (Salmo gairdneri)b Dogfish (Poroderma africanum)b

mGnRH cGnRH 1 cGnRH I1 sGnRH

+ +

+ + + +

+ + + + + +

+ + + +

+

+ + + + + + + + + + +

"Determined by amino acid composition andor sequence analysis. "Determined on the basis of immunological and chromatographic studies and (in most cases) assessment of LH-releasing activity in a chicken pituitary cell bioassay.

GnRH immunoreactivity is also present in the extrahypothalamic brain of birds (King and Millar, 1980; Jozsa and Mess, 1982; Sterling and Sharp, 1982; Knight et al., 1983). In ostrich extrahypothalamic brain, the presence of immunoreactive and bioactive cGnRH I and cGnRH 11 has recently been demonstrated (H. Jach, R. C. Powell, R. P. Millar, and J. A. King, unpublished). 2. Reptiles Immunoreactive GnRH material in extracts of lizard (Mabuya capensis) and tortoise (Chersine angulata) hypothalami exhibited immunological and charge (cation-exchange chromatography) properties different from those of mGnRH and similar to those of chicken and teleost GnRHs (King and

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Millar, 1979a, 1980).The data pointed to alterations in the vicinity of Leu7 of mGnRH. Since GnRH in reptile species is less positively charged than mGnRH, as in cGnRH I (King and Millar, 1979a, 1980), it appears that Arg' is also not present in the GnRHs of these reptiles. More extensive immunological and chromatographic studies have demonstrated that the major form of lizard (Cordylis nigra) brain GnRH is identical to salmon brain GnRH, Trp7, Leu'-GnRH (sGnRH) (Table I) (Fig. 1). The peptide coeluted with sGnRH in a cation-exchange and three reversed-phase HPLC systems, which were specifically designed to separate the four known natural vertebrate GnRHs and a range of GnRH analogs (Powell et al., 1985). The interaction of this immunoreactive peptide with antisera directed against different regions of mGnRH, cGnRH I, and sGnRH was similar to the relative interaction of sGnRH with these antisera. The peptide also had LH-releasing activity similar to that of sGnRH in a chicken pituitary cell bioassay (Powell et al., 1985). In addition, three structurally related GnRH molecular forms were detected. One of these had HPLC properties and chicken LH-releasing activity identical to that of cGnRH 11. In lizard (Podarcis sicula sicula) brain, three GnRH forms have been demonstrated. One of these had immunological and HPLC properties, and LH-releasing activity in a chicken pituitary cell bioassay, identical to sGnRH. The other two are novel GnRH forms, which exhibit some cross-reaction with several GnRH antisera, but cannot be identified as any of the known GnRHs. These peptides also had chicken LH-releasing activity (J. A. King, R. C. Powell, G. Ciarcia, and R. P. Millar, unpublished) (Table 1). The presence of cGnRH I1 has been demonstrated in skink (Calcides ocellatus tiligugu) brain, using chromatographic, immunological, and biological techniques (R. C. Powell, G. Ciarcia, R. P. Millar, and J. A. King, unpublished) (Table I). Alligator (Alligator mississippiensis) hypothalamic GnRH was thought to differ from mGnRH in position eight on the basis of immunological data (Lance, 1985). Recent immunological and high-resolution HPLC chromatography studies on alligator brain extracts have indicated the presence of two GnRH molecular forms, these being identical to cGnRH I and cGnRH I1 (R. C. Powell, V. Lance, R. P. Millar, and J. A. King, unpublished) (Table I). The alligator brain GnRHs also had LH-releasing activities identical to those of cGnRH I and cGnRH I1 in a chicken pituitary cell bioassay. GnRH is also present in extrahypothalamic areas of the reptile brain (King and Millar, 1980; reviewed by Peter, 1983). The regional distribution of the different GnRH types within the reptile brain has not been investigated, but it is likely that specific GnRHs are confined to specific localities within the brain.

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3. Amphibians Hypothalamic GnRH in frogs (Rana pipiens and Rana catesbeianu) (Alpert et al., 1976; Jan et al., 1979; Branton et al., 1982; Eiden et al., 1982)and Xenopus laevis (Deery, 1974; King and Millar, 1979a. 1980)and in the toad (Bufogariepensis) (King and Millar, 1980) was shown to have identical physicochemical properties to that of mGnRH. Purification of frog (R. catesbeiana) brain GnRH revealed a single species with an amino acid composition identical to that of mGnRH (Rivier et al., 1981) (Table I). In X . laevis brain, two forms of GnRH, which have identical chromatographic and immunological properties to mGnRH and cGnRH 11, have been identified (J. A. King and R. P. Millar, unpublished) (Table I). These peptides also had LH-releasing activities similar to those of mGnRH and cGnRH I1 in a chicken pituitary cell bioassay. Hypothalamic immunoreactive GnRH content in X . laevis has been shown to vary in relation to season and reproductive physiological state (King and Millar, 1979b), with high concentrations in reproductively active frogs collected in the breeding season and with low levels in sexually quiescent frogs collected in the nonbreeding season. The role of the two forms of GnRH found in the frog brain in amphibian reproduction has not been determined. Immunological and HPLC studies have revealed that frog (R. catesbeiana) retinal extracts have the mammalian type of peptide in addition to a more hydrophobic species, which has HPLC properties similar to sGnRH and which is thought to differ from mGnRH at Arg' (Eiden et ul., 1982). This additional species of GnRH is the major form found in the frog sympathetic ganglion (Jan et al., 1979; Eiden and Eskay, 1980; Eiden et al., 1982) and adrenal gland (Eiden et al., 1982). Branton et al. (1982) have reported that the GnRH form found in ganglion predominated in brain extracts of metamorphic frog (R. catesbeiana) tadpoles, while mGnRH predominated in the brains of postmetamorphic frogs and adults. In X . laevis tadpoles, the radioimmunoassayable GnRH (middle-directed antiserum 1076) had properties similar to mGnRH (King and Millar, 1981b). The GnRH form detected in the ganglion might also have been present, but was not detected by the antiserum used. Brain immunoreactive GnRH content has been shown to increase steadily during metamorphosis, with a rapid rise at postclimax (King and Millar, 1981b). The functions of the two forms of GnRH during metamorphosis remain to be determined. GnRH-like immunoreactivity has been demonstrated in the brain, retina, and sympathetic ganglion of numerous other amphibians (reviewed by Crim and Vigna, 1983; Peter, 1983), but the nature of the GnRH has not been identified.

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4. Fish Teleost fish (tilapia, Sarotherodon mossambicus) brain GnRH was found to differ from mGnRH in the vicinity of Leu’ (King and Millar, 1979a, 1980). The teleost peptide was less positively charged, suggesting the absence of Arg’ (King and Millar, 1980). Similarly, codfish (Gadus morhuu morhua) brain GnRH (Barnett et al., 1982; Jackson and Pan, 1983) and winter flounder (Pseudopleuroncctes americanus) brain GnRH (Idler and Crim, 1985) were shown to be immunologically different from mGnRH. Purification and sequence analysis of salmon (Oncorhynchus keta) brain GnRH (designated sGnRH) revealed the structure Trp’, Leu8-GnRH (Sherwood et al., 1983) (Fig. I). The salmon brain form of GnRH appears to be widespread in teleost fish. In all the species which have thus far been investigated using immunological and chromatographic techniques, the major GnRH molecular form has identical properties to Trp’, Leu*-GnRH, as in salmon brain (Table I). These species include hake (Merluccius capensis) pituitary gland (King and Millar, 1983, tilapia (Tilapia sparrmanii) brain (King and Millar, 1985), Cork julis brain (R. C. Powell, G . Ciarcia, R. P. Millar, and J. A. King, unpublished), codfish brain (Jackson and Pan, 19831, and mullet (Mugil cephalus), milkfish (Chanos chanos), and trout (Salmo gairdneri) brain (Sherwood et a / . , 1984). Multiple forms of GnRH have been described previously in various species of teleost fish (Barnett et al., 1982; Jackson and Pan, 1983; Sherwood er a / . , 1983. 1984; King et a / . , 1984a; Idler and Crim, 1989, although the nature of these molecular variants of GnRH has not been identified. In recent immunological and chromatographic studies, we have demonstrated that, in addition to sGnRH, hake pituitary gland and tilapia brain also contain cGnRH I (King and Millar. 1985) (Table 1). Higher molecular-weightforms of GnRH have been reported in codfish brain (Barnett at al., 1982; Jackson and Pan, 1983) and in winter flounder brain (Idler and Crim, 1985). Amongst elasmobranch fish, dogfish (Poroderma ufricanum) hypothalamic GnRH was shown to differ from mGnRH in the vicinity of Leu’ (King and Millar, 1980). Recently, two forms of GnRH were identified in dogfish ( P . ufricanum) brain, which have identical chromatographic and immunological properties to sGnRH and cGnRH I1 (R. C. Powell, R. P. Millar, and J. A. King, unpublished) (Table I). These GnRHs also had LH-releasing activities identical to those of sGnRH and cGnRH 11 in a chicken pituitary cell bioassay. Immunoreactive GnRHs have also been detected in another dogfish (Squalus acanthias) (Jackson, 1980; Sherwood and Sower, 1985) and in ratfish (Hydrolasus colliei) (Jackson, 1980). In Scyliorhinus canicula brain, immunoreactive sGnRH is reported to be absent (Breton et ul., 1984).

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ROBERT P. MILLAR AND JUDY A. KING

Considering cyclostomes, immunoreactive GnRH has been demonstrated in hagfish (Heptatretus hexatrema) brain (King and Millar, 1980), Pacific hagfish (Eptatretus stouti) brain (Jackson, 1980), Pacific lamprey (Entosphenus tridentata) brain (Crim et al., 1979a), Western brook lamprey (Lampetra richardsoni) brain (Crim el al., 1979b), and landlocked sea lamprey (Petromyzon marinus) brain (Sherwood and Sower, 1985). The molecular nature of GnRHs in these primitive fish has not been identified. GnRH immunoreactivity is present in the extrahypothalamic brain of fish (King and Millar, 1980; reviewed by Crim and Vigna, 1983; Peter, 1983). However, detailed analysis of the nature of GnRH in fish brain (excluding the hypothalamus) has not been undertaken, and it remains uncertain as to whether there is a differential distribution within the brain of the multiple forms of fish GnRH. 5 . Invertebrates and Lower Organisms Immunoreactive and biologically active GnRH has been demonstrated in the hepatopancreas of the grass shrimp (Penaeus monodon) (Wan et al., 1984). The structure of this GnRH-like material has not been ascertained. A substance with GnRH-like biological activity has been reported in extracts of oak (Avena sativa) leaves (Fukushima et al., 1976).

D. GnRH-RELATED MOLECULES In addition to the various forms of GnRH which have been identified in the different vertebrate species, there are several peptides which have a degree of sequence homology with GnRH (Fig. 3). The sequence homGnRH

mGnRH Prolactin

cGnRH 1 cGR P

pGlu' --Pro147-

His

Trp

His

-

Pro

-

-

Ser

y.

Tyr

Gly

Ser .. Gly

-

Leu

Trp - Ser - Tyr

-

Gly

Val .. Trp

pGlu' H-Alal

.

Leu - Gln

-

Pro - Gly

-

1

1

Pro -%r

- Leu - Gln156--

Leu

-

1

Leu .. Arg

-

Pro

Gly1'*NH2

Gln - Pro - Gly1'*NH2]

Glya J S e r -

Pro

-

Alal'--

FIG. 3. Structural homologies of yeast a-mating factor and mammalian prolactin with mGnRH. and chicken gastrin-releasing peptide (cGRP) with cGnRH 1. "Denoted as being homologous, since the amide of mGnRH (and presumably cGnRH I) is derived from an additional Gly at the COOH-terminus of the mGnRH precursor (see Fig. 2).

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mology of yeast a-mating factor with mGnRH is sufficient to allow specific binding to rat pituitary GnRH receptors and the stimulation of LH release from cultured gonadotrophs, albeit at relatively high concentrations (Loumaye et d . , 1982). 111. Biological Activity of GnRH

A. INTERSPECIFIC GONADOTROPIN-RELEASING ACTIVITIESOF VERTEBRATEGnRHs

Synthetic ovine-porcine GnRH is biologically active at low doses in a wide variety of domestic and laboratory mammals and also in feral mammalian species, including rock hyrax, impala, blesbok, Soay sheep, and hyena (Millar and Aehnelt, 1977; Lincoln, 1979; Illius et al., 1983), suggesting that the GnRH structure and the GnRH receptor have been conserved throughout the class Mammalia. cGnRH I has low gonadotropin-releasing activity using sheep pituitary cells in v i m (Millar and King, 1983a) and in the rat in vivo (Sandow e? al., 1978) (Table 11) (Fig. 4). sGnRH is slightly more active using rat pituitary cells (Sherwood er al., 1983) and sheep pituitary cells (Millar e? al., 1986) in vitro, while cGnRH I1 exhibits a further enhancement in LHreleasing activity in sheep pituitary cells in vitro (MiUar er al., 1986) (Table 11) (Fig. 4). In birds, cGnRH I and mGnRH are equipotent in releasing LH from chicken pituitary cells in vitro, with low ED,,s of 3 x lo-" M (Millar and King, 1983a) (Table 11) (Fig. 4). They are also equipotent in vivo in TABLE II INTERSPECIFIC GONADOTROPIN-RELEASING ACTIVITIES OF VERTEBRATE GnRHs" GnRH type Mammalian Chicken I Chicken II Salmon

Vertebrate class Mammalb

Bird'

100

100 100

Oor 0

600 250

+d

2 8 5

Reptile'

?

+

Amphibianc

Fish

100 100

100

7

100

100 100 100

"Activity of the peptides is shown as a percentage of the activity of mGnRH, except for the reptile studies where 0 indicates no activity and + indicates activity. "ln vitro pituitary cell LH response. 'In vivo bioassay. "o-Arg6-cGnRH I I .

164

ROBERT P. MILLAR A N D JUDY A. KING

I

I

lo-'' 10"

I

I

I

I O - ~ lo-'

PEPTIDE (M)

I

I

lo5

1

10"

1

1

loi11 0 "

I

PEPTIDE (MI

,

(

lo7

FIG.4. LH-releasing activity of mammalian GnRH (-), chicken GnRH I (-.-.-.-). chicken GnRH I1 (----), and salmon GnRH (............) in sheep pituitary cells ( A ) and in chicken pituitary cells (B). Data were adapted from Millar and King (l983a) and from unpublished results.

chickens (Johnson et al., 1984; Sterling and Sharp, 1984) and in quail (Chan et a/., 1983). The two peptides have identical affinities for chicken pituitary GnRH receptors (Millar and King, 1983a). cGnRH I1 is 5.6 times more potent than cGnRH I in releasing LH from chicken pituitary cells (Fig. 4) and 13.5 times more potent in releasing FSH (Millar et al., 1986). sGnRH is 2.5 times more potent than cGnRH I in releasing L H (Fig. 4) and 1.8 times more potent in releasing FSH (Millar et a / . , 1986). In turtles and snakes, mGnRH and cGnRH I are apparently inactive (Licht et al., 1984). In contrast, other studies reported some ability of mGnRH to stimulate the reproductive system in turtles (Callard and Lance, 1977; Licht, 1980). In male alligators, mGnRH was shown t o stimulate plasma testosterone (Lance et al., 1986). Administration of DArg'-cGnRH I1 to female iguanas stimulated plasma estradiol, which ellicited reproductive behavior in males (Phillips et al., 1985) (Table 11). Among amphibians, mGnRH (i.e., one of the frog GnRH forms) stimulates gonadotropic hormone secretion, steroidogenesis, and spawning in anurans (McCreery et al., 1982; Licht et al., 1984). mGnRH has been reported to stimulate spermatogenesis and ovulation in newts (Mazzi et al., 1974; Vellano et al., 1974). cGnRH I is equipotent with mGnRH in stimulating gonadotropin secretion in frogs in vivo (Licht et a / ., 1984) (Table 11). The doses required to achieve these effects are considerably higher than those required to induce gonadotropin release in rats, despite the fact that one of the natural hypothalamic hormones in frogs has the structure of mGnRH. A lower affinity of mGnRH for frog pituitary receptors and/or more rapid degradation of mGnRH may account for this relatively

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

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poor activity. We have observed very rapid degradation of mGnRH by X . laevis plasma (R. P. Millar, unpublished). The sensitivity of frog pituitaries to GnRH in vitro suggests that the receptor-binding affinity is high (P. Licht, personal communication). mGnRH and its analogs have relatively poor gonadotropin-releasing activity in fish when compared with doses required in mammals (Ball, 1981; Crim et al,, 1981; Donaldson et al., 1981; King and Millar, 1981~;Peter, 1983; L. W. Crim, 1984). The structural characterization of salmon brain GnRH as Trp’, Leu*-GnRH (sGnRH) (Sherwood et al., 1983) suggested that this might be due to interclass specificity of GnRH. However, mGnRH and sGnRH are equally effective in stimulating plasma testosterone and 17-P-estradiol in tilapia in vivo (King et al., 1984b), and the peptides are equipotent in stimulating gonadotropin release from superfused goldfish pituitary cells (MacKenzie et al., 1984). All three peptides (mGnRH, cGnRH I , and sGnRH) are equipotent in stimulating gonadotropin secretion in goldfish (Peter et al., 1985) and salmon (L. W. Crim, personal communication) in vivo (Table 11). These observations of interspecific differences in gonadotropin-releasing activities of vertebrate GnRHs (Table 11) emphasize the high specificity of the mammalian GnRH receptor and a relative nonspecificity (“promiscuity”) of the pituitary GnRH receptor in the nonmammalian vertebrates. This question has been addressed in more detail by testing gonadotropin-releasing activity of synthetic GnRH analogs with substitutions in positions five, seven, and eight, which vary among natural vertebrate GnRHs (see below). Aside from GnRH action in regulating pituitary gonadotropin secretion, the peptide(s) appear to act as a neurotransmitter and directly affect reproductive behavior in rats (Moss and McCann, 1973; Pfaff, 1973; Moss et d.,1979). The specificity of these actions has not been thoroughly investigated, although Ac-GnRH (5-10) enhances lordotic behavior in female rats while GnRH (I-6)-NH2 is ineffective (Dudley et d.,1983). Evidence that another form of GnRH exists in mammalian brain (as found in the pineal gland) may indicate that these neurotransmitter effects are not mediated by mGnRH, but by a different form of the peptide. The presence of GnRH receptors in extrapituitary tissues in mammals, such as the testis, ovary, placenta, and adrenal gland (see references in Section II.B,3, and in human mammary carcinoma tissue (Miller et al., 1985; Eidne et al., 1985b)and the biological effects of GnRH and analogs on these tissues point to local sources of GnRH-related peptides which affect these tissues. In view of the evidence for a local production of these GnRH-like peptides, a paracrine or autocrine regulatory system appears to pertain. The specificity and molecular size of gonadal GnRH receptors

166

ROBERT P. MILLAR A N D JUDY A. KING

suggests that they are identical to the pituitary receptor (Reeves et al., 1980), although other studies have found some differences in binding specificity (Perrin et al., 1980; Millar et al., 1982). Evidence has been presented which indicates that testicular GnRH differs structurally from mGnRH (Dutlow and Millar, 1981; Paul1 et al., 1981). However, cGnRH I has a low relative activity in inhibiting gonadal steroidogenesis, similar to its activity in the pituitary (R. P. Millar and A. J. Hsueh, unpublished). Sequence analysis of human placental GnRH mRNA has confirmed an identity with the hypothalamic peptide (Seeburg and Adelman, 1984). mGnRH stimulates human p-chorionic gonadotropin secretion by human placenta in vitro (Belisle et af., 1984), but comparative studies of the effects of other vertebrate GnRHs have not been undertaken. Studies on extrapituitary biological actions of GnRHs in nonmammalian vertebrates are even more limited. In birds, studies have demonstrated a direct effect of mGnRH on gonadal steroidogenesis (Hertelendy et al., 1982) and behavior (Cheng, 1977). mGnRH and sGnRH stimulate nerve firing in bullfrog spinal ganglia (Jan et al., 1980; Jan and Jan, 1983). A GnRH with properties similar to sGnRH has been extracted from this tissue (Eiden et a / . , 1982) and is thought to resemble an amphibian sympathetic neurotransmitter (Jones et al., 1984). mGnRH was 10-fold more potent than sGnRH in pressor activity in the toad (Wilson, 1985). The amating factor in yeast, which has structural homology with mGnRH (Fig. 3), stimulates fusion of gametes, indicating an early evolution of GnRHrelated peptides in regulating reproduction. B. STRUCTURE-ACTIVITY RELATIONSOF GnRH FOR GONADOTROPIN RELEASE

I . Structural Significance of Position Eight Amino Acid Arg in position eight of mGnRH appears to be essential for gonadotropinreleasing activity in mammals. A conservative substitution with Lys reduces biological activity from 10 to 25% (Sandow et al., 1978; Milton et al., 1983). Substitution of Arg' with Gln, Leu, Om, His, and Cit results in low LH-releasing activity in the rat in vivo (Sandow et a f . , 1978), as well as from sheep pituitary cells in culture (R.P. Millar, J. A. King, and R. W. Roeske, unpublished). Of the neutral amino acids, Phe, Trp, Cit, Met, and Leu retain the most LH-releasing activity (5-9%), while Ser, Ile, and Asn have 0.2-2% relative activity (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). Acidic amino acid substitution (e.g., Glu) results in the lowest activity (0.04%). These findings are in accordance with the concept that His', Tyr', and Arg' form a combined unit of hy-

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

I67

drogen bonding important for stabilizing the molecule for biological activity (Shinitzky and Fridkin, 1976). We have proposed that this interaction limits the potential number of GnRH conformers and favors the occurrence of a "preferred" conformer which interacts with the mammalian GnRH receptor (Milton et al., 1983). This relative limitation on conformers is indicated by the narrow titration range (< 1.74 pH units) of His', as monitored in Trp3 fluorescence in the native mGnRH, and also in Lys'GnRH, which retains substantial biological activity. Neutral amino acid substitution results in a titration range of > 1.74 pH units, reflecting a heterogeneous population of His residues in the poorly active analogs, and presumably a greater heterogeneity of GnRH conformers (Milton et al., 1983). cGnRH I falls into this group and has a relative potency of 1-5% in mammalian systems (Sandow et al., 1978; Millar and King, 1983a; Milton et al., 1983). In the bird, these structural requirements of the receptor clearly do not pertain, as cGnRH I is equipotent with mGnRH in chickens (Johnson et al., 1984; Sterling and Sharp, 1984) and quail (Chan et al., 1983) in vivo and in chicken pituitary cells in vitro (Millar and King, 1983a; Milton et al., 1983). Using chicken pituitary membranes, these biological effects were shown to be directly related to receptor-binding affinity (Millar and King, 1983a). We have now defined the requirements of the chicken pituitary GnRH receptor in more detail by comparing the ability of a range of position eight-substituted GnRH analogs to release LH from dispersed chicken pituitary cells. Relative to cGnRH I, Arg' and Phe8 analogs have full LH-releasing activity. Met', His', and Leu' analogs exhibit about 30% activity. Ser', Trp', Cit', and Ile' analogs have 1&20% activity; and only the acidic residue Glu' had low LH-releasing activity (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). Thus, accepting the validity of the structural conformer stabilization model for the mammal, one must conclude that the avian receptor is promiscuous and binds a number of GnRH conformers present in the unstabilized analogs which have substitutions for Arg'. However, this proposal does not exclude the possibility that the importance of a basic amino acid in position eight of GnRH for biological activity is simply related to a charge interaction with a negative charge at the binding site of the mammalian GnRH receptor. Studies on the influence of the position eight amino acid on gonadotropin-releasing activity in reptiles, amphibians, and fish are much less extensive. As indicated above, cGnRH I is active in amphibians and fish (Licht et al., 1984; Peter et al., 1985), cGnRH I1 in certain reptiles (Phillips et al., 1983, and sGnRH is active in fish (King et al., 1984b; MacKenzie et al., 1984; Peter et al., 1985) (Table 11). Although these findings are not sufficiently extensive to draw definite

168

ROBERT P. MILLAR AND JUDY A . KING

conclusions regarding the requirements for the amino acid in position eight in these nonmammalian vertebrates, they do indicate that the pituitary GnRH receptor in these classes is similar to that of the bird in tolerating considerable amino acid variation in this position.

2. Effects of Conformational Constraint on GnRH Bioactivity In support of the postulate that Args plays a role in stabilizing GnRH conformation and that this is important for receptor binding and biological activity in mammals, Freidinger et al. (1980) reported that a GnRH analog containing a y-lactam bridge (between the C of Gly6 and N of Leu7) which stabilizes the (3-turn of residues five to eight of GnRH, was 9 times more potent than native GnRH in stimulating LH release from dispersed rat pituitary cells. This analog is also 9-10 times more active in sheep pituitary cells (R. P. Millar, J. A. King, R. W. Roeske, unpublished). On the basis of the hypothesis that the avian receptor does not require Arg' for conformational stabilization and will bind a number of GnRH conformers, it follows that the y-lactam conformational restraint would not enhance bioactivity in the chicken bioassay. In a recent study, we observed that the stimulation of LH release from dispersed chicken pituitary cells by native mGnRH and the y-lactam analog over the dose range of 10-11-10-6M was indistinguishable (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). These findings, therefore, support the concept that conformational stabilization is less important for GnRH interaction with its receptor in the chicken and quail, and probably in birds in general. The effect of this conformational constraint on GnRH activity in other nonmammalian vertebrates has not been established. 3. Influence of Positions Five and Seven on Biological Activity Studies in mammalian bioassays established that some modifications such as N-methyl-Leu7 actually enhance activity (Ling and Vale, 1979, presumably through stabilizing the (3-turn of GnRH (Chandrasekaren et al., 1973). Ile7-, Nle7-, Ser7-, and (Boc)Lys7-substituted mGnRH analogs retain significant activity in mammalian systems (Sandow et al., 1978), suggesting a degree of nonspecificity of the mammalian receptor for the amino acid in this position. Trp7-mGnRH has high activity when compared with the parent compound (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished) and the presence of Trp7 in cGnRH I1 and sGnRH appears to enhance binding to the mammalian receptor when compared with cGnRH 1. The mammalian receptor is, however, not totally indiscriminate in binding position seven-substituted analogs, as Gly7, Ala7, Val7, Lys7,

EVOLUTION OF GONADOTROPIN-RELEASING H O R M O N E

169

Arg7, D-L~u'.and Pro7 substitution leads to a marked decline in activity (Sandow et al., 1978). The mammalian GnRH receptor appears to differ from the chicken GnRH receptor in its requirements for the amino acid in position five. His5enhances biological activity in the chicken and mammalian bioassays when incorporated in cGnRH 11. This substitution in mGnRH results in an increase in activity in the mammal, while a marked decrease in activity ensues in the chicken (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished). Ala' and Pro' substitution results in reduced gonadotropinreleasing activity in the mammal, while Phe'-mGnRH retains substantial activity (Sandow et ul., 1978). Our observation that sGnRH (Trp7,L e d mGnRH) is 2.5-fold more active than cGnRH I (Gln8-mGnRH) in stimulating LH release from chicken pituitary cells indicates that the avian receptor is also tolerant of alterations in position seven of GnRH. However, it is clear that the combination of substitutions in positions seven and eight can be important as Gln7, Leu8-mGnRH has only 4% relative potency in the chicken pituitary cell system (King et al., 1983). As LeunmGnRH has 30% relative activity, it appears that Gln in position seven is largely contributory to the decline in activity. cGnRH 11 (His', Trp7, Tyr8-mGnRH) is more active than cGnRH I and mGnRH in stimulating gonadotropin release from dispersed chicken pituitary cells (Millar et al., 1986). Since sGnRH, which shares Trp7 in common with cGnRH 11, was also more active than cGnRH I , this residue appears to be responsible for the enhancement in activity. Indeed, Trp7mGnRH is 2.8 times more active than mGnRH in the chicken system (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished). It also appears that His substitution for Tyr' is acceptable to the chicken receptor and may even contribute to the enhanced activity of cGnRH 11. However, the nature of other residues in positions seven and eight is clearly important since His'-mGnRH was actually much less active than any of the natural GnRHs, and His'. Trp7-mGnRH was less active than cGnRH 11 (R. P. Millar, J . A. King, and R. C. deL. Milton, unpublished). Comprehensive data on the influence of amino acids five and seven for GnRH activity in other nonmammalian vertebrates are lacking. The high biological activity of all the naturally occurring vertebrate GnRHs suggests a considerable tolerance of amino acid substitutions in these positions.

4. Structural Requirements for Superactive GnRH Agonists It is now well established that substitution of D-amino acids for Gly", N-methyl-Leu for Leu7, and N-ethylamide for Gly"' - NH2 in GnRH enhances gonadotropin-releasing activity of the peptide (Sandow et d., 1978)

I70

ROBERT P. MILLAR A N D JUDY A. KING

(Fig. 5). In view of the less basic nature and different hydrophobicity of both forms of chicken GnRH and sGnRH and the different requirements of the nonmammalian vertebrates’ pituitary GnRH receptors, the same principles for producing agonists with enhanced activity need not necessarily apply. a. Analogs of Mammalian GnRH. D-Trp6-mGnRH exhibits an enhancement of 26-fold in stimulating L H release from chicken pituitary cells, which is similar to the 36-fold increased activity in sheep pituitary cells (Millar and King, 1983b). o-Ser6(Bu‘),Prog-NHEt-mGnRH,which has enhanced activity in mammals, exhibits increased activity in chickens in vivo (Sterling and Sharp, 1984). On the other hand, ~-His~(Bzl),Pro’-NHEtmGnRH, which has a relative potency of about 50 in the mammal, exhibits only a 4-fold enhancement in the chicken system (R. P. Millar and J. A. King, unpublished). Similarly D-Leu6-mGnRH, which is 27 times more active than mGnRH in releasing L H from rat pituitary cells, has no enhanced activity in chicken pituitary cells (Hasegawa et d . , 1984). A new GnRH agonist, ~-Glu~(anisole),Pro~-NHEt-mGnRH (J. E. Rivier and H. Anderson, unpublished), also displayed no enhancement in activity in chicken pituitary cells in contrast to a 4-fold increase in activity in the sheep pituitary cell bioassay. Similarly D - ~ A ~ ~ ~ ( E ~ , ) , P I - o ’ - N H E ~ - ~ G ~ R H , which is a very active analog in the rat, is only twice a s active as mGnRH in the chicken pituitary cell bioassay (J. J. Nestor and R. P. Millar, unpublished). It is clear, therefore, that the enhanced activity of mGnRH agonists in mammalian systems is Frequently not paralleled by their activity in the chicken. This is likely to be due to the differences in GnRH receptors described earlier and may be related to the fact that conformational constraint of GnRH does not affect biological activity in the chicken system to the same extent as in the mammal. Fewer data on comparative potencies of mGnRH agonists in other nonmammalian vertebrates are available. ~-His~(Im-Bzl),Pro’-NHEt-rnGnRH is 45 times more potent than mGnRH in the bullfrog (McCreery et a f . ,

AGONIST

pGlul

ANTAGONIST

.\“:“i 1

D-AMINO ACID (ESPECIALLY AROMATICS)

-

His2

-

Trp3

1 1

D-AMINO ACID (ESPECIALLY BULKY AROMATICS)

-

Ser4

-

Tyr5

-

Gly

-

Leu7

ETHYLAMIDE

- Arg8 -

D-AMINO ACID (ESPECIALLY BASICS)

Fa. 5. Structure of GnRH agonists and antagonists.

Pro9

-

1

Glylo*NH2

.c

D-Ala

EVOLUTION OF GONADOTROPIN-RELEASING HORMONE

171

1982) in accordance with mammalian data. This analog is approximately 4 times more active than mGnRH in the goldfish (Peter et al., 1985), which is similar to our observations in the chicken (R. P. Millar and J. A. King, unpublished). In turtles, this analog does not increase plasma gonadotropins or steroid levels (Licht et al., 1982). In the frog, D-Ser"(Bu'),Pro9NHET-mGnRH has been shown to stimulate testicular steroidogenesis both in vivo and in vitro (Pierantoni et a l . , 1984). ~-Ala",pro'-NHEtmGnRH, which is 14 times more active than mGnRH in the rat, is active in the African catfish (de Leeuw et al., 1985),but does not exhibit enhanced activity in the goldfish (Peter et al., 1985). Other information on the actions of GnRH analogs in lower vertebrates has been reviewed (L. W. Crim, 1984). b. Analogs ofChicken GnRH I . D-T~~"-cG~RH I is 26 times more potent than the parent peptide in the chicken pituitary cell bioassay (Millar and King, 1983b), which is identical to the increased activity when ~ - T r p "is incorporated in mGnRH. This analog is 100 times more potent than cGnRH I in the sheep pituitary cell bioassay (R. P. Millar and J. A. King, unpublished). In goldfish, D-Trp"-cGnRH I exhibited only a 5-fold increase in activity (Peter et al., 1985), suggesting a difference in the GnRH receptor in birds and fish. However, the analog was approximately 20 times more active than the parent peptide in trout (L. W. Crim, personal communication). D-hArg"(Et,)-cGnRH I exhibited a 5-fold increase in activity in the chicken pituitary cell bioassay (J. J. Nestor and R. P. Millar, unpublished). c. Analogs of Chicken GnRH ZZ. D-Arg" incorporation in cGnRH I1 led to a 4-fold increase in activity in the chicken pituitary cell bioassay (Millar rt al., 1986). D-hArg"(Et,) resulted in no enhancement in the chicken system, but increased the activity 46-fold in the sheep pituitary system when compared with the poor activity of the parent peptide (J. J. Nestor and R. P. Millar, unpublished). The D-Arg" analog appears to exhibit good activity in the iguana (Phillips et al., 1985). d. Analogs of Salmon GnRH. D-Arg", Pro9-NHEt-sGnRH had an 8fold increase in activity in chicken pituitary cells (R. P. Millar, J. E. Rivier, and J. A. King, unpublished) and a 12-fold increase in the goldfish (Peter rt ul., 1985). D-His" (Im-Bzl), Pro9-NHEt-sGnRH was also 8 times more active in the chicken system, but exhibited no significant increase in activity in the goldfish as did a D-Ala" analog (Peter et al., 1985). In summary, it is apparent that the majority of GnRH analogs which are superactive agonists in mammals exhibits relatively little or no increase in activity in the chicken and probably also the other nonmammalian vertebrates. This emphasizes the differences in structural requirements of the respective receptors. Differences in metabolic clearance may con-

I72

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tribute in the in vivo studies, but are unlikely to be of much significance in the in vitro bioassays. Although there is a much closer parallelism of activity in birds and fish, there may also be differences in these receptors, as certain analogs (e.g., DHis6 (Im-Bzl), Pro9-NHEt-sGnRH)have marked differences in activity in the chicken and goldfish bioassays. The studies on GnRH analog activity in the chicken pituitary cell bioassay provide the most comprehensive data and demonstrate that, in general, substitution of a particular D-amino acid for Gly6 results in a similar increase in activity when substituted in any of the naturally occurring vertebrate GnRHs. An exception to this is D-hArg6(Et,) substitution in sGnRH, which is considerably more active than the corresponding mGnRH, cGnRH I, and cGnRH 11 analogs. With the limited information available, it is not possible to formulate definite rules concerning the types of GnRH analogs likely to exhibit enhanced activity in nonmammalian vertebrates, as has emerged for analogs in mammals. However, the sequence of activity of the D-hArg6(Et,)analogs (sGnRH-A > cGnRH 11-A > cGnRH I-A > mGnRH-A) relates to the relative hydrophobicity of these analogs, as has been demonstrated for GnRH analogs in mammals (Nestor et al., 1984).

5 . Structural Requirements for GnRH Antagonists GnRH analogs which have antagonist activity in mammals (Fig. 5 ) were tested for their ability to inhibit the LH responses of sheep and chicken pituitary cells to cGnRH (J. A. King and R. P. Millar, unpublished). The analogs were also tested alone for their intrinsic LH-releasing activity. The most potent antagonist in the chicken pituitary bioassay was Ac~-Phe',~-pCl-Phe~,D-Trp-'.~,D-Ala'~-mGnRH with an IC,, of 5 x lo-' M and no intrinsic LH-releasing activity. In the sheep pituitary cell bioassay, this analog was intermediate in activity with an IC,, of 2.5 x M. ~-pGlu',~-Phe',~-Trp'.~-mGnRH had slightly lower antagonist activity in the chicken system while, it was a very weak antagonist in the sheep had intermediate anbioassay. Ac-~-pCl-Phe'~~,~-Trp~~~,~-Ala'~-mGnRH tagonist activity in both bioassays, and ~ - P h e * , ~ - T r p ' , ~ - P had h e similar ~ activity in the chicken bioassay, but was a weak antagonist in the sheep. The most active antagonist in the sheep bioassay was Ac-D-pCI-Phe'.',DTrp',~-Phe~,~-Ala''-mGnRH (IC,, lo-'' M ) . This antagonist had the weakest antagonist activity in the chicken bioassay (IC,,, M ) and displayed agonistic activity when administered alone, with an ED,,, of M. There are, therefore, marked differences in the antagonistic and agonistic activities of these analogs in the chicken and sheep bioassays, which fur-

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ther indicates differences in the receptors with respect to their GnRH structural requirements for interaction with the NH2-terminal part of the molecule. Differences in this regard are perhaps unexpected, as the sequence of the NH,-terminal region of GnRH (amino acids 1-4) has been completely conserved in vertebrate GnRHs, and a similar conservation of the receptor in its interaction with this region might have been anticipated. An antagonist incorporating the natural Trp7of cGnRH I1 and sGnRH rather than the Leu7 of rnGnRH has been shown to have increased potency in rats (Folkers et al., 1984). Among lower vertebrates, a mGnRH antagonist (Ac-dehydro-Pro',pCI~-Phe',~-Trp',~,NaMeLeu~-mGnRH) has been shown to block mGnRHinduced gonadotropin release in the bullfrog (McCreery et al., 1982). DPhe2,Phe3,D-Pheb-mGnRHinhibits mGnRH-stimulated gonadotropin release in trout (Crim et al., 1981). 6. Pituitary Desensitization Since pituitary desensitization to prolonged and/or high doses of GnRH is a receptor-mediated event in the rat (Smith and Conn, 1984), it was of interest to determine the influence of the differences in the nonrnammalian receptor on expression of desensitization. When dispersed chicken pituitary cells suspended in a Biogel column were stimulated with 2-minute pulses of M cGnRH I every 30 minutes for 3% hours, a LH response was associated with every pulse. In contrast, a definite desensitization of the pituitary cells occurred when they were continuously perifused with M cGnRH I or 10-7M D-Trp6-cGnRHI (Millar and King, 1984). After 100 minutes of perifusion, LH release declined to basal levels. In view of our difficulty in satisfactorily quantitating GnRH receptors in chicken pituitary cells or membranes (Millar and King, 1983a),we have been unable to determine whether the receptor "down-regulation" plays a major part in pituitary desensitization, as in the rat (Zilberstein et al., 1983). In chickens in vivo, daily injections of D - S ~ ~ ~ ( B U ' ) , P ~ ~ ~ - N H E ~ - ~ G ~ did not reduce pituitary responsiveness to the analog (Sterling and Sharp, have ~ - ~been ~ G ~reRH 1984). Prolonged doses of D - S ~ ~ ~ ( B U ' ) , P ~ O ~ - N H E ported to induce desensitization in lizards in vivo (Ciarcia et al., 1983). while in turtles this desensitization phenomenon has not been observed (Licht et al., 1982). The perifused bullfrog pituitary continues responding to sustained high doses of GnRH and lacks the phenomenon of desensitization (McCreery and Licht, 1983). The goldfish pituitary is reported to exhibit desensitization when exposed to superactive GnRH analogs (Peter, 1980).

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IV. Conclusions

It is evident that heterogeneous molecular forms of GnRH are present within vertebrates. GnRH structure also varies in different tissues within the same vertebrate species and even within the same tissue, as occurs in the brains of several nonmammalian vertebrate species. At present, four forms of GnRH have been structurally characterized: the original GnRH from porcine and ovine hypothalamus; Gln8-GnRH and His’,Trp7,Tyr8-GnRHin chicken hypothalamus; and Trp7,Leu8-GnRHin salmon brain. Immunological, chromatographic, and biological properties indicate the presence of other forms of GnRH in mammalian and nonmammalian tissues which await characterization. All of the GnRHs exhibit gonadotropin-releasing activity, especially in nonmammalian vertebrates in which the pituitary receptor is relatively nondiscriminatory in binding GnRHs with substitutions in positions five, seven, and eight. However, it is conceivable that the GnRHs have other biological activities, such as is demonstrated by the stimulation of growth hormone secretion in fish, actions on the placenta, ovary, testis, and adrenal gland in certain mammals, and effects in the central and peripheral nervous systems. It appears, therefore, that the basic GnRH structure has been recruited through evolutionary selective processes to serve diverse functions. The specificity of these actions is achieved in a variety of ways: (1) through the evolution of different molecular forms of GnRH, which interact with tissue-specific receptors; (2) through paracrine regulation by anatomically closely localized cells, which does not allow the peptide to enter the general circulation in concentrations sufficient to bind other GnRH receptors. In this system, which appears to pertain in the gonads, the GnRH and receptors in different tissues can be identical; (3) through the existence of a “private” conducting system, as is found in the hypothalamohypophyseal portal system; and (4) through intimate contact between the secretory and target cells, as occurs in neuronal communication. In addition to the heterogeneity of GnRHs within vertebrates and in tissues of the same species, it is apparent that at least two forms of the peptide are generally present in the hypothalamus (or brain) of nonmammalian vertebrates. In mammals, only a single molecular form has been isolated, and only a single GnRH sequence has been detected in the mammalian genome (Seeburg and Adelman, 1984). However, the presence of a different form of GnRH in the mammalian pineal gland suggests that two or more forms are present in the mammalian brain (and possibly hypothalamus). Conceivably, the detection of only one GnRH-coding sequence in the mammalian genome may be due to significant structural

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differences in other form(s) which do not hybridize well with the mGnRH probe. The functional significance of two hypothalamic GnRHs in vertebrates has not been established. In the chicken, both forms of hypothalamic GnRH stimulate pituitary secretion of LH and FSH at concentrations appropriate for physiological regulation (
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erance of amino acid substitutions in position eight, it is evident that they are not identical in this regard and display distinct differences. The mammalian pituitary GnRH receptor shows some similarity to the nonmammalian receptors in its tolerance of substitutions in positions five and seven. Distinct differences do exist, however, as is evident in the acceptance of His’ substitution for activity in the mammal, while this change causes a decline in activity in the chicken. The conservation of the NH,-terminal sequence of GnRH during evolution suggests that this part of the molecule plays a vital role in interacting with the receptor. Appropriate amino acid substitutions in this region produce molecules which still bind the receptor, but do not activate gonadotropin release (antagonists) (Sandow, 1982). Cross-linking these antagonists by antibodies reestablishes gonadotropin release (Conn et al., 1982; Gregory et al., 1982), suggesting that the structural information in the NH,-terminus of GnRH is to do with receptor aggregation required for initiating intracellular biochemical events which mediate gonadotropin release. There is some indication that the avian receptor domain, which interacts with the NHI-terminal region of GnRH, may also differ from that of the mammal in that a series of GnRH antagonists exhibited different properties in mammalian and chicken bioassay systems. The mammalian and avian GnRH receptors also differ in molecular weight. Using a ligandimmunoblotting technique (Eidne et al., 1985a), we recently demonstrated a binding protein for GnRH in chicken pituitary membranes which was -7,000 Da larger than the well-established 60,000-Da mammalian pituitary GnRH receptor or receptor-binding subunit. The presence of two forms of GnRH in representative species of the major vertebrate classes suggests that gene duplication occurred early in vertebrate evolution or even preceded the earliest vertebrates. Consideration of the nucleotide-coding sequences for the known vertebrate GnRHs reveals that cGnRH I1 is the most different from the other GnRHs. as a minimum of three nucleotide changes are required to account for the amino acid changes. Since cGnRH I1 is also the most commonly represented form in vertebrates, it may have arisen early after duplication of the gene and been highly conserved during evolution in a similar way to vasotocin (Acher, 1983). A single base change is sufficient to account for an interchange between cGnRH I and mGnRH and a double base change for cGnRH I cf sGnRH and mGnRH cf sGnRH. It is possible, therefore, that these forms represent the other evolutionary arm of the duplicated gene in which there has been greater diversity of structures akin to that of the oxytocin-like peptides, which comprise some six known structures in vertebrates (Acher, 1983).The distinctly different activities of oxytocin and vasopressin reemphasize the possibility that some of the “GnRHs”

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may have functions unrelated to the stimulation of gonadotropin release. Nevertheless, GnRH may have a very ancient evolutionary origin as a regulator of reproduction in view of the structural similarity between mGnRH and the yeast a-mating factor. The multiplicity of actions of GnRHs in reproduction, where they act in the central nervous system to stimulate reproductive behavior, in the pituitary to stimulate gonadotropic hormones, in the gonad to affect steroidogenesis, in mammary carcinoma cells to affect growth, and in the placenta to affect human chorionic gonadotropin secretion, appears to be a remarkable conservation of function within a major physiological system. However, it is apparent that GnRHlike sequences are present in other peptides with nonreproductive functions. A sequence in chicken gastrin-releasing peptide has close homology with cGnRH I, and sequences having homology with mGnRH occur in prolactin (Fig. 3). There appears to be considerable plasticity in the cooption of certain peptide sequences for diverse functions during the course of evolution.

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