Structure and evolution of GnRH

GnRH is a single-chain decapeptide produced in the hypothalamus. Its amino acid sequence was first established in 1971 (Matsuo et al., 1971b, Matsuo et al., 1971a, Burgus et al., 1972) thereby confirming the existence of a hypothalamic factor stimulating the release of both LH and FSH ftrom the anterior pituitary, as had been predicted as early as 1947 (Green and Harris, 1947). The sequence for mammalian GnRH is shown in figure 1.5.

The sequence of hypothalamic GnRH (GnRH-1) is highly conserved between mammalian species, but varies up to 50% between other vertebrate species (Kasten et al., 1996). Despite this variation its function as a regulator of reproduction has been conserved during 500 million years of evolution (Millar and King, 1988).

1 2 3 4 5 6 7 8 9 10

pGlu His Trp Ser Tyr Gly Leu A r g Pro Gly-NH2 mam m a l i a n GnRH I pGlii His Trp Ser His Gly Trp Tyr Pro Gly-NH2 m a m m a l i a n GnRH II pGl u His Trp Ser Tyr Gly Trp Leu Pro Gly-NH2 m a m m a l i a n GnRH III

p G l u His Trp Ser Tyr Gly Leu Gin Pro Gly-NH2 c hicken GnRH I p G l u His Trp Ser Tyr Gly Trp Leu Pro Gly-NH2 salmon GnRH

pGl u His Tyr Ser Leu Glu Trp Lys Pro Gly-NH2 lamprey GnRH

GnRH agonists :

Leuprolide pGlu His Trp Ser Tyr DLeu Leu Arg Pro NHEt Buserelin pGlu His Trp Ser Tyr DSer (O^Bu) Leu A r g Pro NHEt Nafarelin pGlu His Trp Ser Tyr 02NalLeu Arg Pro Gly-NH2 Gosrelin pGlu His Trp Ser Tyr DSer (O^Bu) Leu Arg Pro AzaglyNH2 Histrelin pGlu His Trp Ser Tyr DHis(Bzl)Leu Arg Pro AzaglyNH2 Triptorelin pamoate pGlu His Trp Ser Tyr DTrp Leu Arg Pro Gly-NH2

GnRH antagonists:

Nal-Glu Antagonist Nal-Glu, ( [Ac-D2Nal\D4ClPhe2,D3Pal^,Arg\ DGlu^(AA)

Cetrolix [N-Ac-D-Nal (2) S D-Phe (pCl) D-Pal (3) \ D-Cit®, D-Ala^°] GnRH Ganirelix [N-Ac-D-Nal (2) \ D-pCl-Phe2,D-Pal (3) \ D-Arg (Et) ^ (8) , D-Ala^°] GnRH Detirelix [N-Ac-D-Nal (2) \ D-pCl-Phe^, D-Trp\ DhArg (Et2 ) o-Ala^°] GnRH

Figure 1.5 GnRH sequences

a) The amino acid sequences of six GnRH isoforms.

There are three known mammalian forms. The boxed parts of the sequences are the most invariant parts, i.e. the C- and N-termini. The N terminus is post-translationaliy modified to contain a pyro-glutamic acid and the C-terminus has been amidated.

b) GnRH agonist and antagonists

Some GnRH agonists that are currently available for clinical use and some GnRH antagonists that are undergoing clinical trials. GnRH agonists and antagonists are hoped to be of general use in treatment of prostate cancer in men and possibly also as a means of fertility control, given along with testosterone. (Based on figure in Yen, 1999)

General Introduction

It is now believed that there are at least three different forms of GnRH: GnRH-I, GnRH-II and GnRH-III (see figure 1.5). GnRH-I, the hypothalamic form, has a major role in reproductive regulation but its sequence varies greatly (up to 50%) between species (Sherwood et al., 1993). It is not only localised in the central nervous system, although it is in highest concentrations there, but also in the placenta, pancreas, gonads and some tumour cell lines (Sherwood et al., 1993). GnRH-II was first isolated fi*om chicken brain and found to have His in position 6, Trp in position 7 and Tyr in position 8. (Miyamoto et al., 1984). It is invariant and has been found in such diverse species

as fish, birds and mammals. GnRH-II is believed to be the most ancient and conserved form of GnRH (Millar and King, 1988).

The human gene encoding GnRH-II has recently been isolated and characterised (White et al., 1998). Both GnRH-I and II genes have four exons and encode a precursor containing signal sequences preceding the GnRH decapeptide, a three amino acid proteolytic site and a gonadotrophin-associated peptide (GAP, see below). This is the general organisation of the GnRH-I and GnRH-II genes in all other species analysed (Sherwood et al., 1993). The two GnRH precursors differ in size due to differences in the GAP sequence, which is 50% longer in GnRH-II than in GnRH-I (White et al., 1998). The main site for GnRH-II expression is in the extrahypothalamic areas of the midbrain (Chen et al., 1998) and it is also found in high levels in the kidney, bone marrow and prostate (White et al., 1998).

A third form of GnRH (GnRH-III) is found in the telencephalon of several fish species (Sherwood et al., 1983, Sherwood et al., 1984). A recent study showed that a related GnRH-III is expressed in the hypothalamic and midbrain neurones in mammals, including humans (Yahalom et al., 1999).

The biological functions of the different isoforms o f GnRH are not yet clear. GnRH-II can act as a neuromodulator in spinal cord ganglion neurones in amphibia (Jan et al., 1980, Muske et al., 1994). Differentiated lymphocytes can also express GnRH or a GnRH-like peptide (Rissman et al., 1995, Marchetti et al., 1996). The diversity in their gene expression patterns would indicate that the GnRH peptides have diverse neurosecretory, endocrinological and even immunological roles.

AU studied vertebrate GnRHs have an effect on the reproductive system, by stimulating gonadotrophin release. But the effects vary as there are considerable differences in the receptor binding and gonadotrophin-releasing activity o f the different forms of GnRH (Millar et al., 1989, Millar et al., 1986). GnRH receptors have been found throughout the body, e.g. in the kidney and the prostate (Kakar and Jennes, 1995, Fekete et al., 1989). Recently a GnRH receptor specific for GnRH-II has been found in primates. It differs slightly in structure to the GnRH-I receptor and the two receptors are only 41% homologous in their nucleotide sequences, but they are both expressed ubiquitously in the body (Neill et al., 2001, Millar et al., 2001).

As shown in figure 1.5 the amino- and carboxyl-terminal ends of GnRH are highly conserved in vertebrates. Experiments with GnRH-analogues have shown that their N- and C-terminal sequences are involved in receptor binding and activation (Millar and King, 1988). GnRH has a bend in its middle region (Tyr^-Gly^-Leu^-Arg*) that brings the N- and C-termini close together in its active form (Momany, 1976, Lincoln, 1992). The N-terminus of mammalian GnRH is also protected by cyclization of the N- terminal glutamine to pyro-glutamic acid and the C-terminus is amidated (Lincoln,

1992, Pal et al., 1996).

Since GnRH-I is the predominant form of GnRH in the hypothalamus and the pituitary portal vessels (Chen et al., 1998) and is the most efUcient of the known GnRH forms at stimulating LH release, in subsequent chapters ‘GnRH’ will refer to mammalian GnRH-I, unless otherwise stated.

GnRH agonists and antagonists

Numerous analogues of GnRH have been synthesised for possible therapeutic application. The responses to these GnRH-analogues have been evaluated by various different methods: LH secretion from pituitary cells in vitro, ovulation induction in vivo, oestrus suppression and inhibition of FSH-induced oestrogen production by granulosa cells in vitro (Hsueh et al, 1983). The analogues can be divided into agonist and antagonists. Agonists are more stable than the native form of GnRH and therefore have a longer half-life. They also have a higher receptor affinity, which along with the longer half-life results in increased biologic activity. Increased stability of the peptide is achieved for example by substitution at position 6 by a D-amino acid. Increased

General Introduction

receptor affinity is found by replacement of the C-terminal glycinamide residue with ethylamide. The interaction of GnRH with its receptor is discussed in detail in section 1.7.3. Some of these agonists are 200 times more efficient at stimulating the release of gonadotrophins from the pituitary than the native form of GnRH (Yen, 1999). The initial stimulation is however followed by down-regulation of the gonadotrophin- gonadal axis (see section 1.7.3).

Some synthetic antagonists on the other hand have higher affinity to the GnRH receptor than native GnRH, but do not evoke receptor activation. They are normally modified at the C- and N-termini or have deletions or substitutions at positions 2 and 3 (Karten and Rivier, 1986). A few agonists and antagonists of GnRH are hsted in figure 1.5.