Download Structure–function relationship of follicle

 European Society for Human Reproduction and Embryology
Human Reproduction Update 1998, Vol. 4, No. 3 pp. 260–283
Structure–function relationship of
follicle-stimulating hormone and its receptor
Alfredo Ulloa-Aguirre1,3 and Carlos Timossi2
1Department of
Reproductive Biology, Instituto Nacional de la Nutrición SZ, México D.F. and 2Department of Pharmacology,
Faculty of Medicine, Universidad Nacional Autónoma de México, México D.F.
TABLE OF CONTENTS
Introduction
Structure of FSH
FSH receptor structure and function
Structural determinants for subunit assembly,
FSH–FSH receptor interaction and signal
transduction
Conclusions
Acknowledgements
References
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Key words: FSH/FSH receptor/function/structure
Introduction
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Follicle stimulating hormone (FSH) is one of the two
pituitary gonadotrophins involved in the regulation of
gonadal function. Structurally, this gonadotrophin is a
heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in
both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors,
also displays a high degree of functional and molecular
complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural
determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity
and diversity that allows formation of distinct ligandreceptor complexes capable of selectively activating or
deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and
functional epitopes for intra- and extracellular hormone action are of paramount importance not only for
a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function
3To
but also for the rational design of analogues with predicted properties and effects.
Cells communicate with each other through chemical signals. These signalling molecules bind to specific receptors
located in the target cell. Upon binding, the signalling molecule activates receptor proteins, which in turn transduce
the signal carried by the specific extracellular messengers.
The activation of such receptor proteins initiates a cascade
of events that are amplified at each level and that eventually
culminate in a highly sensitive and specific cellular response. The particular structural features of a given signal
determine not only its metabolic fate in the circulation but
also its ability to bind and activate a specific target cell
receptor, to generate a signal transduction and to induce a
biological effect.
Follicle stimulating hormone (FSH), one of the master
signals produced by the anterior pituitary gland, is involved
in the regulation of several essential reproductive processes
occurring at the gonadal level (Chappel et al., 1983a). This
gonadotrophin belongs to a family of closely related glycoproteins composed by FSH, luteinizing hormone (LH), choriogonadotrophin hormone (CG) and thyroid stimulating
hormone (TSH), which are synthesized in different cell
types; TSH is synthesized in a distinct pituitary cell, LH and
FSH are synthesized by the gonadotropes and CG is produced by the placental trophoblasts. All these glycoprotein
hormones are heterodimers consisting of a common α-subunit non-covalently associated with a β-subunit, which is
structurally unique in its peptide sequence to each member of
the family (Pierce and Parsons, 1981; Gharib et al., 1990b).
In fact, within a given animal species, the α-subunits arise
from a single gene whereas the β-subunits arise from separ-
whom correspondence should be addressed at: Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de
Quiroga No. 15, Col. Tlalpan, C.P. 14000, México D.F., México. Tel: (525) 573–11–60; Fax: (525) 655–9859; Email: [email protected].
FSH and FSH receptor
ate genes and confer to each hormone a high degree of
biochemical and biological specificity (Godine et al., 1982;
Fiddes and Talmadge, 1984; Gordon et al., 1988; Mercer and
Chin, 1995). Although the specificity of each hormone resides in the β-subunit, there is ample evidence suggesting
that in fact both subunits contact the receptor (Lapthorn et
al., 1994; Zeng et al., 1995; Dias, 1996; Liu and Dias, 1996;
Remy et al., 1996; Grossman et al., 1997). The subunits of
these glycoprotein hormones contain one or two Asn-linked
(N-linked) heterogeneous oligosaccharides which have been
shown to play an important role in both the in-vivo and
in-vitro bioactivity of the hormone (Baenziger and Green,
1988; Stockell and Renwich, 1992; Smith et al., 1990).
Moreover, within a single hormone, the wide variability in
oligosaccharide composition constitutes the main chemical
basis for isoform formation and the large molecular heterogeneity exhibited by each hormone (Chappel et al., 1983a;
Ulloa-Aguirre et al., 1988a; Ulloa-Aguirre et al., 1995).
Whereas TSH and FSH bind to distinct receptors, LH and
human chorionic gonadotrophin (HCG) share the same receptor (Combarnous, 1992; Vassart and Dumont, 1992; Segaloff and Ascoli, 1993; Simoni et al., 1997). Nevertheless,
receptors for all these trophic hormones belong to the superfamily of G-protein-coupled receptors and thus share several
major structural features (Ulloa-Aguirre and Conn, 1998).
Granulosa cells and Sertoli cells are the target cells for
FSH action and they are the only cell types which express
the FSH receptor (Simoni et al., 1997). In the ovary, FSH
regulates granulosa cell function, including the development and selection of ovarian follicles, oocyte maturation
and, in concert with LH, the ovulatory process. In the
testes, FSH regulates the function of Sertoli cells, which in
turn provide physical and biochemical support for proper
development and maturation of germ cells. Binding of the
FSH molecule to and activation of its cognate receptor
involves the participation of several structural determinants of the ligand present in both the primary sequence
and the carbohydrate residues of the molecule (Combarnous, 1992). Characterization of the molecular basis of
FSH–FSH receptor interaction and signal transduction is of
paramount importance from a clinical point of view since it
may allow the development and production of a variety of
analogues potentially useful to exogenously regulate gonadal function.
This review concentrates on the role of specific structural
determinants present in the FSH molecule and its cognate
receptor important for their function and interaction. Special
emphasis is placed on the importance of naturally occurring
variations in the oligosaccharide structure of FSH in determining the biological activity of the hormone.
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Structure of FSH
As mentioned above, FSH is a heterodimer composed of
two glycosylated dissimilar subunits, α and β (Pierce and
Parsons, 1981; Ward et al., 1991). The common α-subunit
gene is coordinately expressed with the different β-subunit
genes and its hormonal control varies in a cell-type specific
manner (Kay and Jameson, 1992; Schoderbeck et al.,
1992, 1993; Pennathur et al., 1993; Kim et al., 1994; BenMenahem et al., 1995). In contrast, the β-subunit genes are
restrictively expressed and exhibit a more limited and specific hormonal regulation (Papavasiliou et al., 1986; Dalkin et al., 1989; Steger et al., 1993; Steinfelder and
Wondisford, 1997). Both subunits are non-covalently associated and apparently individual subunits are biologically
inactive. The fact that each β-subunit is able to combine
with the common α-subunit (Pierce et al., 1971), suggests
that some regions in the three-dimensional structure of the
β-subunits may be very similar among the different glycoprotein hormones. Thus, the variable regions are probably
those which confer immunological and biological specificities to the hormone.
The common α-subunit
The gene encoding the common α-subunit of several mammalian species consists of four exons separated by three
intervening introns (Naylor et al., 1983; Gharib et al.,
1990b). In humans, the gene is located on chromosome 6
(Naylor et al., 1983); although a high degree of conservation in introns position and sequence homology is present
among different species, particularly mammals, the size of
the gene presents considerable variations attributable to
differences in the first intron (Gharib et al., 1990b). In
general, the α-subunit mRNA varies between 730 and 800
nucleotides; it encodes for a leader sequence of 24 amino
acids followed by the sequence of the mature protein,
which is composed of 92 amino acid residues in humans
and 96 residues in other mammalian species (Boothby et
al., 1981; Godine et al., 1982; Naylor et al., 1983; Gordon
et al., 1988; Gharib et al., 1990b) (Figure 1). Based on its
corresponding gene structure, the α-subunit may be divided into three separate regions or domains (Fiddes and
Talmadge, 1984). The first domain, comprised of the
amino acid residues 1 to 10, shows the highest degree of
interspecies variability (Fiddes and Talmadge, 1984; Ryan
et al., 1987). In the human α-subunit, the absence of four
amino acids corresponding to residues 6 to 9 in other
species produces a gap in the comparative Cys-aligned
sequence of the subunit (Chin et al., 1983); however, this
missing sequence does not alter the extension of any of the
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A.Ulloa-Aguirre and C.Timossi
Figure 1. The sequence of the human common α-subunit (hFSHα; upper panel) and hFSHβ subunit (lower panel). Solid brackets indicate the
position of the disulphide bridges (according to Pierce and Parsons, 1981; Ryan et al., 1987; Bousfield et al., 1994; Lapthorn et al., 1994).
Branched structures indicate the position of N-linked glycosylation sites.
disulphide bridges of the subunit (Bousfield et al., 1994).
This is the only gap that has been detected in mammals.
The extensive degree of sequence heterogeneity found in
the NH2-terminal end of the α-subunit (Keutmann et al.,
1978) suggests that this domain is not involved in the biological activity or dimer assembly of the hormone. The
other two domains (residues 11 to 71 and 72 to 96) exhibit a
high degree of homology (70–90%) among mammals
(Ryan et al., 1987).
The best-characterized mechanism for transcriptional
activation of the α-subunit gene is through the cAMP/protein kinase A (PKA) pathway (Jameson et al., 1986a). In
the human, the α-subunit gene promoter exhibits tandemly
repeated cAMP/PKA response elements (CRE) which are
located at positions –146 to –111 relative to the transcription initiation site (Jameson et al., 1986a). The CRE consist
of the conserved palindromic sequence TGACGTCA. This
motif is found in many genes regulated by cAMP and it
binds the nuclear factor CREB (CRE-binding protein),
activating gene transcription (Deutsch et al., 1988b). Protein kinase C (PKC) and calcium-calmodulin protein kinases also modify CREB (Deutsch et al., 1988a,b) and
activate transcription of the α-subunit gene. On the other
hand, oestrogens inhibit FSHα-subunit synthesis by a
mechanism that is independent of oestrogen receptor binding to the subunit promoter (Keri et al., 1991). This is in
contrast to the FSHβ gene which contains a high-affinity
binding site for oestrogen receptor in its 5’ flanking region
(Kim et al., 1988)
The presence of disuphide bonds confers a highly folding three-dimensional structure to the subunit and they are
essential to maintain the biological activity of the
α/β-dimer (Fujiki et al., 1980). Formation of disuphide
bonds begins during the synthesis of the polypetide (cotranslationally) and probably concludes after α/β assembly
in the rough endoplasmic reticulum (Huth et al., 1993).
The signal peptide also exhibits a half-cysteine which is not
oxidized to the disuphide form prior to its co-translational
removal from the mature protein. Half-cysteine locations
are highly conserved among the α-subunits of several
species indicating that their tertiary structure is very similar
(Bousfield et al., 1994). In humans, the common α-subunit
bears 10 cysteine residues that form five intrachain disuphide bridges (Lapthorn et al., 1994) (Figure 1). Among
these bridges, those that bind Cys residues at positions
α10–60, α28–82 and α32–84 are involved in the so-called
cysteine knot (Lapthorn et al., 1994). This cysteine knot
motif is also found in HCGβ and presumptively in the
β-subunits of the other glycoprotein hormones; it is apparently composed of two disuphide bonds that form a ring
through which a third bridge passes (McDonald and Hendrickson, 1993; Lapthorn et al., 1994). Although the
α-subunit of human heterodimeric glycoprotein hormones
exhibits nearly the same conformation, subtle differences
which depend on the particular β-subunit present, have
been detected (Dighe et al., 1990). On the other hand,
disruption of disuphide bonds either at position 7–31 or
59–87 in the human α-subunit markedly reduces heterodimerization with the LHβ-subunit without affecting its
assembly to FSHβ or HCGβ (Furuhashi et al., 1996). This
observation suggests that regions in the α-subunit recognized by a specific β-subunit for the heterodimer assembly
FSH and FSH receptor
may differ among these gonadotrophins. Apparently, Nlinked oligosaccharides on the α-subunit play an important
role in the folding of different functional domains of the
subunit and probably affect the flexibility of the molecule
which is critical for subunit assembly and possibly disuphide bridge formation (Matzuk and Boime, 1988a,
1989).
Glycosylation of the subunits of glycoprotein hormones
begins in the rough endoplasmic reticulum (RER) with the
co-translational
transfer
of
dolichol-linked
Glc3Man9GlcNAc2 oligosaccharide precursor to asparagines at glycosylation consensus sites Asn-X-Ser/Thr
(Baenziger and Green, 1988). In the glycoprotein hormones, N-linked oligosaccharides exhibit a common core
consisting of two N-acetylglucosamine and three mannose
residues (Baenzinger and Green, 1988) (Figure 2.). After
dimer formation and trimming of glucose and mannose
residues to a Man5GlcNAc2 in the RER and cis-Golgi,
extensive processing of the oligosaccharides attached to
the protein core of the hormone occurs in the medial and
trans-Golgi to form the mature oligosaccharides (Bousfield et al., 1994, 1996). The numerous carbohydrate intermediates resulting from this post-translational processing,
several of which become final forms of the complex
carbohydrate chains attached to the protein core, are responsible for many of the gonadotrophin glycoforms synthesized and secreted by the pituitary (Baenziger and
Green, 1988; Stockell and Renwich, 1992; Ulloa-Aguirre
et al., 1995; Chappel, 1995) (Figure 2). The majority of
Asn-linked oligosaccharides in FSH are mono-, di- and
trisialylated bianntenary structures, which confer an overall negative charge to the molecule (Ulloa-Aguirre et al.,
1995). The human α-subunit contains two N-linked oligosaccharides at positions Asn52 and Asn78. As discussed
below, only the oligosaccharide attached to αAsn52 is essential for the biological activity of the α/βdimer (Valove et
al., 1994). Although it has been found that glycosylation of
HCGα in this Asn52 position plays a critical role in dimer
secretion (Matzuk and Boime, 1988a, 1989), deletion of
this residue from FSH by site-directed mutagenesis does
not apparently affect the secretory behaviour of the molecule (Bishop et al., 1994; Flack et al., 1994b; Valove et al.,
1994). In contrast to the α-subunit present in α/β-dimers,
the free α-subunit fraction produced by the placenta and
bovine pituitary bears an O-linked oligosaccharide attached in Thr39/43 (Parsons et al., 1983); although this
additional O-linked oligosaccharide prevents binding of
the subunit to the β-subunit (Parsons and Pierce, 1984) it
does not interfere with its regulated secretion by the gonadotrope (Boime et al., 1982). The regulation of the remarkably different distribution of sialylated and sulphated
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Figure 2. Some of the N-linked oligosaccharide structures found on
ovine, bovine, and human pituitary follicle stimulating hormone.
Only the complete structures are represented. Many other oligosaccharides may be found that are incomplete versions of these structures, mainly lacking terminal residues such as sulphate, sialic acid,
and fucose.
oligosaccharides in LH and FSH are of particular interest
considering that both are synthesized in the same cell (Herbert, 1975, 1976; Baenzinger and Green, 1988). Processing
of the oligosaccharides linked to the α/β-dimer depends on
the specific β-subunit associated. In fact, an N-acetyl galactosamine transferase has been identified in pituitary
membranes that recognizes a tripeptide motif (Pro-X-ArgLys) in the α-subunit forming an LHα/β-dimer, adding a
GalNac residue to its oligosaccharides and leading to a
terminal sulphated sequence (Smith and Baenzinger,
1988). In contrast, FSH oligosaccharides terminate predominantly in a galactose-sialic acid sequence thus indicating that the different patterns of glycosylation for FSH and
LH are modulated by its associated β-subunit. Apparently,
the presence of distinct oligosaccharides in FSH and LH
direct both gonadotrophins into separated secretory granules and/or pathways (Smith and Baenzinger, 1988;
Muyan et al., 1994).
The FSHβ-subunit
In humans, the β-subunit of FSH (FSHβ) is encoded by a
single gene located in chromosome 11 (Watkins et al.,
1987). All mammalian FSHβ subunits have 111 amino
acids and both the nucleotide and amino acid sequences are
highly conserved among species (Maurer and Beck, 1986;
Watkins et al., 1987; Gharib et al., 1989). The nucleotide
sequence of the FSHβ gene predicts an 18–19 amino acid
signal sequence (Jameson et al., 1988) (Figure 1). The
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A.Ulloa-Aguirre and C.Timossi
FSHβ gene is similar to those of other pituitary glycoprotein hormones (Kim et al., 1988; Gharib et al., 1989). It is
comprised of three exons and two introns. Analogous to the
TSH β-subunit gene, the first intron of FSHβ is located six
base pairs upstream from the start of translation (Jameson
et al., 1988). The first exon contains the 5’ untranslated
region while the second and third exons contain all the
coding sequence. The second intron is located between the
sequence coding for amino acids 35 and 36, a strictly conserved position among all the glycoprotein β-subunit genes
(Jameson et al., 1988). However, the FSHβ gene differs
from the other glycoprotein hormone β subunits in that it
possesses an extremely long 3’ terminal and encoded by
the third exon (Jameson et al., 1988; Kim et al., 1988).
Although the biological significance of this long 3’ untranslated sequence in FSH is still unknown, in other proteins, such as in the β-globin mRNA, the sequence
destabilizes its mRNA thereby increasing its turnover rate
(Shaw and Kamen, 1986; Weiss et al., 1995). The transcription initiation site is located 63 nucleotides and the
TATA box 94 nucleotides upstream of the first intron
boundary (Jameson et al., 1988). FSHβ expresses at least
four mRNA transcripts in normal pituitary cells and two
transcripts in neoplastic cells (Jameson et al., 1986b,
1988). There is a lack of similarity between the FSHβ and
LHβ 5’ flanking regions. In fact, a palindromic sequence
in the 5’ flanking region of the FSHβ gene
(GGTCANNNTGACC), that could operate as an oestrogen-responsive element, differs from its homologous 5’
region in the LHβ gene (GGACACCATCTGTCC) (Shupnik et al., 1989b). Despite abundant evidence indicating
that the FSHβ gene is regulated at the level of gene expression, little is known about the exact molecular mechanisms
involved (Albanese et al., 1996).
FSHβ is internally linked by six disulphide bonds (Figure 1). Although establishment of the exact positioning of
the disuphide bridges has proven to be difficult, the most
reliable positions in human FSHβ are those between halfcystines 87–94, 20–104 and perhaps 17–66 (Pierce and
Parsons, 1981; Ryan et al., 1987). The location of these
disulphide bonds agrees with those of their counterparts in
HCGβ as deducted from its crystal structure (Lapthorn et
al., 1994). The exact position of the remaining three disulphide bridges has also been difficult to ascertain mainly
because the reduction–reoxidation reactions necessary to
determine the location of the bridges may produce several
intermediate disulphide bonds during refolding (Ryan et
al., 1987). Different disulphide bridge positions, between
amino acids 3–84, 28–82 and 32–51 of human FSHβ, have
been proposed by others (Fujiki et al., 1980; Bousfield et
al., 1994). Of these, only the 28–82 bridge is in agreement
with the position of its counterpart in HCGβ (Lapthorn et
al., 1994). Presumably, as deducted from the crystal structure of HCGβ and the comparative Cys-aligned sequence
between both β-subunits, disulphide bonds 3–51, 28–82
and 32–84 form the cysteine knot in human FSHβ. In this
setting, the FSHα/β heterodimer would be stabilized by a
segment of the β-subunit which wraps around the α-subunit and is covalently linked like a seat belt by the disulphide bridge β20–104. More recently, pairs of cystine
residues have been artificially introduced in the α- and
β-subunits of FSH, LH and HCG at positions with optimal
geometries for the formation of disulphide bonds (Heikoop
et al., 1997). The mutants formed intersubunit disuphide
bridges and displayed enhanced thermostability relative to
the corresponding heterodimeric glycoprotein hormones,
rendering them candidates for long-acting gonadotrophins
with improved shelf lives. Furthermore, the mutants displayed receptor binding and biological activity like wildtype FSH (Heikoop et al., 1997).
All human glycoprotein hormones, including FSH, have
a well-characterized Cys28-Ala-Gly-Tyr31 (CAGY) sequence in their β-subunits (Pierce and Parsons, 1981). This
quartet of amino acid residues, encoded by exon 2, is also
present in rat FSHβ with the Ala29 residue replaced by Glu
(Gharib et al., 1989). In HCGβ, the Gly36 residue is essential for the production of this hormone (Azuma et al.,
1990), whereas the Tyr37 residue, albeit not obligatory,
may participate either directly or indirectly in subunit assembly and its hydroxyl group may function as a modulatory factor in intracellular signalling (Xia et al., 1993). In
TSH, the CAGY region of the TSHβ gene is apparently
critical for α/β-dimer formation (Nakamura and Nakao,
1993). The importance and significance of this sequence in
FSHβ, which is also present in some unrelated enzymes
and toxins (Kurosky et al., 1977), is still unknown.
FSHβ includes also two N-linked glycosylation sites; in
humans they are at positions Asn7 and Asn24 (Baenzinger
and Green, 1988; Ulloa-Aguirre et al., 1995). However, in
contrast to the common α-subunit, no O-linked carbohydrates are found in any pituitary human β-subunit. Further,
non-combined pituitary β-subunits are not secreted but
rather retained in the endoplasmic reticulum. As described
above for the α-subunit, oligosaccharides attached to the
β-subunit play a critical role in determining the specific
assembly of the subunits as well as the particular carbohydrate processing that occurs after dimer formation (Matzuk
and Boime, 1988b). In addition, they play a pivotal role in
determining the metabolic clearance pattern of the gonadotrophin (Bishop et al., 1995; see below).
The FSHβ gene is specifically regulated by two dimeric
proteins, inhibin and activin, by a single chain polypeptide,
FSH and FSH receptor
follistatin, and by gonadotrophin-releasing hormone
(GnRH) (Ying, 1988; Albanese et al., 1996; Besecke et al.,
1996). Inhibin, activin, and follistatin are produced by the
gonads as well as by a variety of extragonadal tissues such
as the pituitary and the kidney (all three proteins), the placenta and bone marrow (inhibin and activin), the adrenal
gland and the brain (inhibin), and the hypothalamus
(Mason et al., 1985; Petraglia et al., 1987; Meunier et
al.,1988; Sawcheko et al., 1988; Ying, 1988; Shimasaki et
al., 1989). Since the pituitary has the potential to synthesize
all these primarily gonadal proteins, it is conceivable that
they may act as autocrine agents to modulate FSH production by the pituitary gland. Although inhibin is considered
as a selective suppressor of FSH synthesis and secretion
both in vitro and in vivo (Rivier et al., 1986), it has been
found that this protein may also regulate GnRH-induced
LH output in vitro (Ying, 1988). However, in physiological
conditions, the effects of inhibin are apparently masked by
the presence of the comparatively larger suppressive influence provided by testosterone (Culler, 1990; Culler and
Negro-Vilar, 1990). Activin increases the synthesis of
FSHβ and thus the secretion of FSH, whereas follistatin
acts indirectly by binding to and bioneutralizing the effects
of activin (Krummen et al., 1993; de Winter et al., 1996) as
well as through membrane associations with heparan sulphate chains of proteoglycans favouring activin binding to
its receptors and eventually its uptake into the cells and
degradation by lysosomal enzymes (Sugino et al., 1993;
Hashimoto et al.,1997). It has been shown that pituitary
adenylate cyclase-activating polypeptide may stimulate
α-subunit gene expression and suppress FSHβ mRNA
(Tsujii et al., 1994, 1995); however, the action of this neuropeptide upon FSHβ mRNA is apparently exerted via
stimulation of follistatin gene transcription (Winters et al.,
1997). Although a pulsatile GnRH stimulus is also required
for FSHβ gene expression and FSH secretion, the patterns
of synthesis and secretion of this gonadotrophin diverge
from those of LH, which is also under GnRH control.
Whereas fast GnRH pulse frequencies tend to favour LH
synthesis and secretion, slower GnRH frequencies favour
FSH (Turgeon and Waring, 1982; Dalkin et al., 1989).
Recent studies have suggested that the action of the pulsatile GnRH stimulus upon FSHβ gene expression may be
exerted indirectly through changes in activin and follistatin
tone (Besecke et al., 1996). It has been shown that sex
steroids (oestradiol, testosterone and progesterone) also
regulate FSHβ production and FSH secretion in a variety
of experimental conditions (Winters et al., 1979, 1992;
Winters and Troen, 1985; Gharib et al., 1987); however,
their specific effects on FSHβ mRNA production vary depending on the animal species studied and whether the
265
experiments are performed in vivo or in vitro (Rhea et al.,
1986; Wierman et al., 1989; Wierman and Wang, 1990).
For example, in the ovariectomized sheep oestradiol treatment decreases FSHβ mRNA concentrations (Mercer et
al., 1989), whereas in the rat this sex steroid does not exert
any effect on the transcription of the FSHβ gene, at least in
in-vitro conditions (Shupnik et al., 1989a). In cultured
pituitary cells, testosterone directly increases FSHβ
mRNA concentrations in the absence of GnRH (Gharib et
al., 1990a), whereas in the presence of this gonadotrophinreleasing peptide it inhibits synthesis of all three gonadotrophin subunits (Jakubowiak et al., 1991; Winters et al.,
1992). On the other hand, it has been reported that synthesis of both FSHβ-subunit and FSHβ mRNA remain
unaltered in castrated male rats treated with testosterone
(Gharib et al., 1987; Wierman et al., 1989). Finally, expression of endogenous FSH subunit genes by the gonads
has been recently demonstrated (Markkula et al., 1995,
1996). This finding suggests a paracrine and/or autocrine
regulatory role of FSH in gonadal function.
FSH receptor structure and function
The initial event in the action of glycoprotein hormones is
the binding to highly specific receptors located in the membrane of the target cell (Ji et al., 1995). The receptor of FSH
is a glycoprotein that belongs to the superfamily of G protein-coupled receptors (Simoni et al., 1997; Ulloa-Aguirre
and Conn, 1998). These G protein-linked cell surface receptors mediate their intracellular actions through the activation of one or more guanine-nucleotide-binding
signal-transducing proteins (G proteins). Receptors belonging to this superfamily consist of a single polypeptide
chain of variable length that threads back and forth across
the lipid bilayer seven times forming characteristic α-helical membrane-spanning domains, connected by alternating extracellular and intracellular loops oriented to form a
ligand binding pocket (Baldwin, 1993; Strader et al.,
1994).Topographically, the seven transmembrane (TM)
domains form a barrel shape, oriented roughly perpendicular to the plane of the membrane, the extracellular NH2and the intracellular COOH-terminus, and the three extracellular and intracellularly connecting loops (UlloaAguirre and Conn, 1998). The glycoprotein hormone
receptors for FSH, LH/CG and TSH represent a small subclass of the rhodopsin/β-adrenergic large subfamily, to
which the majority of G protein-linked receptors identified
to date belong (Probst et al., 1992; Misrahi et al., 1993;
Ulloa-Aguirre and Conn, 1998).
Similar to that of the luteinizing hormone/choriogonadotrophin receptor (LH/CG-R), the human FSH receptor
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A.Ulloa-Aguirre and C.Timossi
Figure 3. Structural organization of the human follicle stimulating hormone receptor gene. Exons 1 to 10 are represented by octagons. Numbers
above each octagon indicate the number of base pairs of the corresponding exon. Intron length is indicated by the numbers between exons in kbp.
(FSH-R) gene is localized on chromosome 2p21-p16,
which suggests that these two genes, encoding very closely
related glycoproteins, may be a result of gene duplication
(Rousseau-Merck et al., 1990, 1993; Gromoll et al., 1994).
The human FSH-R gene encodes for 678–695 amino acid
residues (Minegishi et al., 1991; Kelton et al., 1992); the
overall nucleotide sequence identity between the human
testicular FSH receptor and its counterpart in the rat is
∼88%, whereas the amino acid identity is ∼90% (87% in
the extracellular domain, 96% in the TM domain and 79%
in the COOH-terminal domain) (Sprengel et al., 1990;
Minegishi et al., 1991; Heckert et al., 1992; Kelton et al.,
1992). Sequence homology of the human FSH-R with the
human LH/CG-R is ∼50% (45% in the large extracellular
domain, 70% in the TM domain and 61% and 10% in the
NH2-terminus and the COOH-terminus of the intracellular
domain respectively) (Misrahi et al., 1993). In the rat and
the human, the FSH-R gene consists of 10 exons separated
by nine very large introns; the relatively small (69 to 251
bp) first nine exons of the gene code for the large NH2-terminal extracellular domain whereas the single large exon
(1225 to 1234 bp) encodes the seven TM-spanning regions
and the COOH-terminal tail of the protein (Figure 3). The
organization of the FSH-R gene is very similar to that
present in both the TSH and LH/CG receptor genes, again
indicating its common evolutionary origin (Minegishi et
al., 1991; Kelton et al., 1992; Misrahi et al., 1993; Gromoll
et al., 1996a; Richard et al., 1997).
Functional characteristics of the extracellular and
the TM domains
In contrast to other members of the rhodopsin/β-adrenergic
subfamily of G protein-coupled receptors, which exhibit
relatively short extracellular domains, the glycoprotein receptors have large extracellular domains which display to
14 sequence repeats (encoded by exons 2 to 9) built on a
motif similar to other leucine-rich glycoproteins (Braun et
al., 1991; Bhowmick et al., 1996; Thomas et al., 1996)
(Figure 4). In the FSH-R, these leucine-rich repeats (in
particular, composite regions encompassed by repeats
1–11), which favour the formation of amphipathic peptide
surfaces optimal for specific protein–protein interactions,
are involved in high affinity binding and binding specificity. The particular structure of this region and the conformational changes occurring upon recognition of the
β-subunit of the hormone, allow receptor stimulation after
proper orientation and positioning of specific regions located in both subunits of the dimer into a receptor activation site defined by the exoloops and the TM domains of
the receptor (Braun et al., 1991; Ji et al., 1995). Several
lines of evidence support this two-step model for hormone–receptor interaction. Based on the molecular structure of porcine ribonuclease inhibitor, a member of the
leucine-rich repeat protein family, it might be deduced that
the extracellular N-terminal domain of the glycoprotein
receptors represents a collar-like structure on top of the TM
domain; considering this structure, the possible site of interaction of the receptor might be molded by amino acid
residues located on both the extracellular domain and the
TM domain (Braun et al., 1991; Kobe and Deisenhofer,
1995). Studies employing a synthetic peptide corresponding to amino acids 9–30 of the rat FSH-R (a sequence not
present in the LH or the TSH receptors) suggest that FSH
interaction with the receptor involves an initial recognition
of the external portion of the NH2-terminal domain by
multiple and discontinuous regions (residues 1–15, 71–85
and 101–111; see below) of the β-subunit of FSH aligned
linearly in a three-dimensional conformation; after binding, conformational changes in the hormone–receptor
complex may allow binding of additional regions (33–53
and 81–95) on the β-subunit which may interact with other
regions of the receptor to allow optimal orientation of the
hormone molecule into the receptor activation site (Dattatreyamurty and Reichert, 1992, 1993; Reichert, 1994).
FSH and FSH receptor
Similar studies have also indicated that amino acids
201–301 and 300–315 in the same receptor are additionally
involved in specific binding of FSH (Sharma and Catteral,
1995; Leng et al., 1995a). As mentioned above, the hydrophobic TM domain of G protein-coupled receptors, formed
by seven α-helical structures, is oriented as a barrel-shaped
pocket, optimal for small molecule high-affinity binding
(Ulloa-Aguirre and Conn, 1998); within this particular
structure (confirmed for rhodopsin and β2 adrenergic receptors; Findlay and Pappin 1986; Wang et al., 1989;
Schertler et al., 1993; Baldwin, 1993), the hydrophobic
amino acid residues on one side of the helix might interact
with the lipid membrane whereas the hydrophilic residues
are exposed to the inside of the pocket, allowing the interaction with hydrophilic surfaces of the ligands (El Tayar,
1996). According to this model, propagation of signal
transduction may involve conformational changes in the
TM domain triggered by hormone-induced subtle alterations in the exoloops connecting particular helices. In fact,
Ji and Ji (1995) have identified several amino acid residues
located in exoloop 1 of the human FSH-R, that may form
one-turn helix extension from the TM-2, and that are involved differentially in hormone binding (His407) and receptor activation (Asp405, Thr408 and Lys409) (Figure 4).
These and other investigators (Quintana et al., 1993;
Shenker et al., 1993; Gilchrist et al., 1996; Ryu et al., 1996;
Fernandez and Puett, 1996) have also identified specific
amino acid residues within the TM-2 (Asp387), TM-6
(Asp557) and exoloop 3 (Lys583) of the LH/CG-R that are
involved in hormone binding and signal transduction;
given the structural and functional similarities among the
glycoprotein hormone receptors, it is highly possible that
these same residues, which are in positions Asp391 (within
TM-2), Asp564 (in TM-6) and Lys591 (located at the interface of exoloop 3 and TM-7, not exposed but oriented
inward to the central cleft facing Asp564) in the human
FSH-R may also be structural requirements for optimal
ligand-induced receptor activation. Therefore, receptor
binding and activation and the subsequent generation of
second messengers are the final result of a series of complex and dynamic interactions between the hormone and
several regions of its cognate receptor.
The extracellular domain, which as described above is
essential for specific binding to FSH, also contains several
(three or four) consensus sequences (Asn-Xaa-Ser/Thr) for
N-linked glycosylation (Mineghisi et al., 1991; Kelton et
al., 1992); whereas glycosylation of the FSH-R is not involved directly in hormone binding, the receptor strictly
requires at least one N-linked glycosylated site for proper
folding, membrane expression and function (Davis et al.,
1995). This is in contrast to the LH/CG-R in which preven-
267
tion of receptor glycosylation does not significantly affect
its cell-surface expression or binding capacity (Tapanainen
et al., 1993; Rozell et al., 1995; Davis et al., 1997).
Several alternately spliced mRNA transcripts coding for
different truncated variants of the FSH-R have been described (Gromoll et al., 1992; Khan et al., 1993; Sairam et
al., 1996, 1997; Yarney et al., 1997) and some of these
receptor variants may be expressed on the cell surface (Sairam et al., 1996). The structural and functional features of
two recently characterized ovine FSH-R variants are of
particular interest. One of these variants, exhibiting a selective alteration in the carboxyl-terminus domain (as the result of a divergent sequence probably originating from a
segment of the gene located beyond exon 10), behaved as a
dominant negative receptor counteracting the FSH-induced signalling properties of the wild-type receptor
(Yarney et al., 1997). The second variant, lacked the conventional seven transmembrane domain and exhibited a
particular carboxyl-terminus of 36 amino acid residues
containing a putative transmembrane segment; interestingly, some of the structural and functional features of this
particular variant were similar to those exhibited by growth
factor receptors (Sairam et al., 1997). Although it has been
proposed that the expression of truncated or altered isoforms may represent a mechanism through which the cell
regulates and defines the specific type of response to the
FSH signal (Gromoll et al., 1992; Ulloa-Aguirre et al.,
1995; Sairam et al., 1996, 1997), more studies are necessary before assigning a physiological role to such receptor
diversity.
Role of the intracellular domains in G protein
coupling and activation
It has been shown that in the rhodopsin/β-adrenergic subfamily of G protein-linked receptors, the intracellular domains, particularly the regions closest to the plasma
membrane in loops 2 and 3 as well as some specific regions
located in the intracellular ends of the TM helices and in the
membrane-proximal portion of the COOH-terminus, are
important for receptor–G protein coupling, interaction and
specificity determination (Franke et al., 1990; Okamoto
and Nishimoto, 1992). G proteins are heterotrimers individually termed α-, β- and γ-subunits (these two latter subunits are tightly bound to each other and form the so-called
β/γ complex) which are encoded by distinct genes (Sprang,
1997). They are signal-transducing molecules, regulated
by guanine nucleotides, that carry the information received
by the receptor to downstream specific cellular effectors
including enzymes and ion channels. The FSH-R is preferentially coupled to the protein Gs which activates the en-
268
A.Ulloa-Aguirre and C.Timossi
Figure 4. Putative membrane topography of the mature human follicle stimulating hormone receptor (Minegishi et al., 1991) showing some
structural characteristics, including putative glycosylation sites (branched structures) as well as some amino acid residues involved in signal
transduction (square residues), mutations leading to inactivation (shaded residues at positions Ala172 and Phe574) or constitutive activation
(black circle at position Asp550) of the receptor, consensus sequences for phosphorylation by protein kinase-A (squares and rectangles) and
protein kinase-C (oval), and potential sites for palmitoylation (Cys627 and Cys629). Cysteine residues in the extracellular domain are outlined in
bold. Brakets in the COOH end of the third intracellular loop and the NH2-end of the COOH tail delimit the B-B-X-X-B motif reversed which has
been shown to be associated with G protein coupling in other G protein-coupled receptors (Wong et al., 1990; Okamoto and Nishimoto, 1992).
Note that the sequence corresponding to the signal peptide is not included in this topographic representation since this sequence is not present in
the mature cell membrane-expressed receptor. Consensus for phosphorylation by protein kinase A and protein kinase C were deducted from
Kenelly and Krebs (1991) and palmitoylation in Cys residues from Bouvier et al. (1995).
zyme adenylyl cyclase to enhance the synthesis of the
second messenger cAMP, which in turns activates protein
kinase A (PKA) (Reichert and Dattatreyamurty, 1989).
Unlike the rat FSH-R, human LH/CG-R and hTSH-R,
which are also coupled to the protein Gq/11 [associated with
activation of phospholipase-Cβ, which catalyses the hydrolysis of the lipid phosphatidylinositol 4,5-biphosphate
to form two second messengers, inositol 1,4,5-triphosphate
and the protein kinase C (PKC) activator, diacylglycerol],
the human FSH-R is weakly coupled to this G protein-mediated signalling pathway (Hirsch et al., 1996). It should be
noted, however, that the intracellular domains of the
human FSH-R exhibit several, albeit weak, consensus sequences for PKC-mediated receptor phosphorylation
(Kennelly and Krebs, 1991; Minegishi et al., 1991; Kelton
et al., 1992). Agonist-induced phosphorylation of the recombinant rat LH/CG- and FSH-R by PKC has been well
documented (Hipkin et al., 1993; Quintana et al., 1994)
and, more recently, FSH-induced receptor phosphorylation
has been documented in a rat granulosa cell line overexpressing the FSH receptor (Selvaraj and Amsterdam,
1997). Apparently, activation of the PKA pathway is not
necessary for phosphorylation of the rat FSH-R (Quintana
et al., 1994). Interestingly, whereas integrity of two-thirds
of the COOH-terminal segment of the cytoplasmic tail
(which bears at least five consensus sequences for PKCmediated Ser/Thr phosphorylation) is required for agonistor phorbol ester-induced (PKC-dependent) receptor phosphorylation and early desensitization of the rat LH/CG-R
(Wang et al., 1996), in the rat FSH-R the corresponding
region (which exhibits also five consensus sequences for
PKC) is not apparently involved in these regulatory processes (Hipkin et al., 1995), thus implicating other intracellular domains as targets for the factors that trigger
FSH and FSH receptor
receptor desensitization. It remains to be investigated
whether the human FSH counterpart is phosphorylated by
this particular kinase as a mechanism of homologous receptor desensitization. FSH-R coupling to the Gi/o protein
(which inhibits activation of adenylyl cyclase) has been
suggested by several studies. Eskola et al. (1994) explored
the effects of pertussis toxin (PTX; a toxin from Bordetella
pertusis which prevents coupling of the Gi protein with the
receptor impeding adenylyl cyclase inhibition) on FSH-induced cAMP production by testis cells from immature rats;
they found that PTX potentiated the effects of FSH on
cAMP generation. In the sheep, an alternately spliced form
of the testicular FSH-R cDNA identical to the wild-type
receptor except for shortening of the carboxyl terminus and
a different peptide segment and that behaves as a dominant
negative receptor presumably through its coupling to Gi
proteins has been described (Sairam et al., 1996; Yarney et
al., 1997). More recently, it has been shown that insect-cell
expressed human FSH may behave as both an agonist or
antagonist of human pituitary FSH and that its inhibitory
effects may be efficiently blocked by PTX, thus suggesting
receptor coupling to the inhibitory Gi/o-mediated pathway
(Arey et al., 1997). Recent observations indicate that the
LH/CG-R may activate phospholipase Cβ via the
Gβγ-dimer released from Gi (Herrlich et al., 1996; Rajagopalan-Gupta et al., 1997). Whether or not a similar signalling cascade also participates in the effector activation
triggered by the FSH-R–G protein system remains to be
ascertained.
Studies employing synthetic peptides corresponding to
the COOH-terminal portion of the third intracellular loop
(amino acid residues Lys551-Ile-Ala-Lys-Arg555) of the rat
FSH-R, have demonstrated that this particular region is
critically involved in receptor coupling to the Gs protein
and hence in adenylyl cyclase activation, cAMP formation
and 17β-oestradiol synthesis (Grasso et al., 1995a,b). This
receptor region contains a reversed minimal structural
motif (B-X-X-B-B, where B represents a basic residue and
X a non-basic residue) which has been shown to determine
the G protein-activating activity in other G protein-coupled
receptors (Wong et al., 1990; Okamoto and Nishimoto,
1992). Although the human FSH receptor also exhibits the
consensus sequence Arg552-Ile-Ala-Lys-Arg556 in the
COOH end of its third intracellular loop (a B-B-X-X-B
motif reversed), coupling of this human FSH-R sequence
to G proteins has not been yet documented. In fact, the
significance of this particular motif in the third intracellular
loop of the LH/CG-R is controversial since mutations involving the lysine residues of this motif have no effect on
signal transduction. Similar to other members of the rhodopsin/β-adrenergic G protein receptor subfamily, the
269
COOH-terminal domain of the FSH-R is also involved in
G protein activation. However, whereas in the rhodopsin
and β2-adrenergic receptors the region corresponding to
the so-called fourth intracellular loop (formed by palmitoylation of Cys residues located in the membrane-proximal region of the COOH-terminus) is involved in G protein
activation, in the rat FSH receptor a portion more proximal
to the COOH-terminal end, encompassed by amino acid
residues Arg650-Lys-Ser-His653 [B-B-X-B motif (Okamoto and Nishimoto, 1992)], is associated with Gs protein
activation (Grasso et al., 1995a,c). The human FSH-R also
contains a B-B-X-X-B motif reversed (B-X-X-B-B) in the
COOH-terminal tail (residues 614 to 618; Figure 4); however, as with the reversed motif located at the COOH-end
of the third intracellular loop, coupling to G proteins has
not been experimentally corroborated. Although the FSHR has Cys residues (in position 628 in the rat FSH-R and in
positions 627 and 629 in the human counterpart) proximal
to the NH2 end of its intracellular tail, it has not yet been
demonstrated that this receptor presents palmitoylation in
these particular sites. It should be emphasized, however,
that mutation of these highly conserved Cys residues in the
LH/CG-R had no effect on agonist-mediated activation of
its corresponding G proteins (Gs and Gq/11) (Zhu et al.,
1995).
Three mutations (1 activating and 2 inactivating) of the
human FSH receptor have been reported. Gromoll et al.
(1996b) described a patient with an activating mutation of
the human FSH-R which resulted in autonomously sustained spermatogenesis in the absence of gonadotrophins.
A heterozygous A→G base change at nucleotide position
1700 (exon 10), leading to an Asp→Gly substitution at
residue 550 in the third intracytoplasmic loop of the mature
protein, was identified (Figure 4). In the inactivating mutation, a C→T point mutation was detected in nucleotide 566
(exon 7) predicting an Ala→Val substitution at residue 172
of the extracellular domain of the membrane-expressed
human FSH-R (Aittomäki et al., 1995). In females, this
mutation leads to hypergonadotrophic ovarian dysgenesis
due to a dramatic reduction of the receptor that binds FSH
and induce signal transduction (Aittomäki et al., 1995).
Interestingly, males homozygous for this inactivating
FSH-R mutation exhibit variable degrees of spermatogenic
failure but without azoospermia or absolute infertility (Tapanainen et al., 1997). Finally, a missense mutation at nucleotide 1777 of the open reading frame that converts
codon 591 from phenylalanine to serine (at position 574 in
the mature, membrane-expressed receptor) has been described in a subset of sex cord tumours (Kotlar et al., 1997).
Functional studies have suggested that this is an inactivating mutation as opposed to the activating mutations which
270
A.Ulloa-Aguirre and C.Timossi
eventually may lead to neoplastic transformation in other
target tissues for glycoprotein hormones (Parma et al.,
1993).
Structural determinants for subunit assembly,
FSH–FSH receptor interaction and signal
transduction
Role of sequence specific peptides and amino acid
residues
Different experimental approaches have been used to map
the structural determinants involved in FSH function.
These include the use of monoclonal antibodies (Mabs)
and synthetic peptides to localize regions in the molecule
important for subunit–subunit, subunit–receptor and
dimer–receptor interaction and the subsequent signal transduction (Reichert et al., 1991; Dias, 1992; Reichert, 1994).
More recently, site-directed mutagenesis has proved to be a
unique experimental approach to identify in more detail
such key functional determinants (Roth and Dias, 1995;
Grossmann et al., 1996). Although each method has its
inherent advantages and disadvantages, all may be considered to complement each other. The immunological
studies have identified three human α-subunit sequences
(amino acid residues α1–15, 11–27, and 33–58) masked by
FSHβ and located in or in close proximity to the α/β subunit interface, suggesting that they may be involved in
subunit assembly (Weiner et al., 1990). In contrast, the
region corresponding to residues α73–92 and to a lesser
extent the overlapping epitope comprised by residues
61–78 on FSHα are surface-oriented and apparently may
be important for receptor binding (Weiner et al., 1991).
Thus, according to the immunological approach, the first
two-thirds of FSHα appear to be involved in subunit–subunit interaction whereas the last third of the subunit may
participate in hormone–receptor interaction. In addition,
this approach has revealed that other glycoprotein hormones do not share all the epitopes mapped in FSHα; some
of these, the so-called conformationally active (flexible)
regions are unique for heterodimeric FSH whereas others
are common to the four human glycoprotein hormones and
are called conformationally and evolutionarily constrained
(rigid) (Weiner and Dias, 1992). This observation suggests
that FSHβ determines the identity of the FSHα epitopes
and that the receptor-binding site of the α-subunit, although identical at the peptide level among glycoprotein
hormones, may be specifically different for FSH.
FSHβ is highly antigenic, and has a particular conformation that enables all antibody-recognizable regions to be in
close proximity to each other (Vakharia et al., 1990).
Immunological studies on this subunit have shown that the
epitopes for anti-human FSHβ33–53 and anti-human
FSHβ81–100 peptides, the former exhibiting a higher affinity for free FSHβ, are masked or altered in heterodimeric FSH, suggesting that they may have an active role in
subunit–subunit association (Vakharia et al., 1990, 1991).
It has also been shown that the β33–53 sequence may be
additionally involved in receptor binding (Sluss et al.,
1986; Santa-Coloma et al., 1990). Peptide sequences
β49–67 and β66–85 are neighboring sequences to β33–53
in an assembled epitope containing determinants for receptor binding (Vakharia et al., 1990, 1991). In fact, high titres
of antibody against the latter sequence blocked the action
of FSH in the rat ovary, disrupting the estrous cycles (Butterstein et al., 1993).
The use of synthetic peptides has complemented the information yielded by immunological techniques and allowed the exploration in a more functionally oriented
manner of the specific sequences involved in subunit association and receptor activation. These short sequences,
mostly between 10 and 20 amino acids in length, may bind
to the human FSH receptor and either block its ligand-binding site(s) and signal transduction capacity or induce receptor activation as assessed by conventional receptor-binding
assays and in-vitro bioassays (Reichert et al., 1991). Using
this approach, the candidates for hormone–receptor interaction studies stem from the analysis of the structural parameters flexibility, surface probability, secondary structure
prediction (α helices and β loops), hydrophobicity and
hydrophilicity of the intact dimer using specialized computer programmes (Devereux et al., 1984; Santa-Coloma
and Reichert, 1990; Reichert et al., 1991; Wako and Ishii,
1995). However, since the majority of these studies have
been performed employing high concentrations of peptides, their results must be taken with caution. The synthetic peptide strategy has been used to study
structure–function relationships of the common α-subunit.
Peptides α33–58, α51–65 or α61–78, corresponding to a
highly conserved portion of human FSHα, completely inhibited subunit association, whereas peptides corresponding to the remaining regions of the subunit (α1–15,
11–27, 22–39 and 73–92), were unable to inhibit
α/β-dimer formation (Krystek et al., 1992). On the other
hand, Leng et al. (1995b,1996) showed that residues
α32–46 were involved in FSH binding with its cognate
receptor. This study suggested that the specific residues
Phe33, Arg35, Arg42, Ser43 and Lys44 in the α-subunit may
be important, and Cys32 essential for human FSH binding.
Interestingly, it has been found that high doses of three
synthetic α-subunit peptides corresponding to α1–15,
α30–45 and α71–85 sequences may bind the LH/HCG
receptor and stimulate testosterone production by rat Ley-
FSH and FSH receptor
dig cells (Erickson et al., 1990). Whether or not these regions participate in FSH receptor activation remains to be
elucidated.
The synthetic peptides corresponding to sequences
31–45, 33–53, 81–95 and 81–100 of human FSHβ inhibited FSH binding to receptor whereas peptides β33–53,
β81–95 and β81–100 stimulated basal levels of oestradiol
synthesis by cultured rat Sertoli cells when tested at high
doses (Santa-Coloma and Reichert, 1990; Santa-Coloma et
al., 1990; Vakharia et al., 1991; Dias, 1992). In agreement
with immunological studies (Vakharia et al., 1990), the
β33–53 and 81–95 sequences are apparently close to each
other in the secondary structure of the subunit forming a
receptor-binding surface of the hormone (Santa-Coloma et
al., 1991a). In fact, a synthetic peptide encompassing both
peptides (FSHβ33–53/81–95) has higher affinity for the
FSH receptor than each peptide alone (Santa-Coloma et al.,
1991a). The overall findings suggest that each of these
binding components effectively interact with the receptor,
providing evidence that these two separate receptor-binding regions in human FSHβ may be forming a discontinuous binding surface in the native molecule. Interestingly,
these FSHβ peptides display thioredoxin-like activity,
which, in the presence of free sulphydryl groups from the
FSH receptor, may induce disulphide interchange and
formation of high-affinity ligand–receptor complexes
(Santa-Coloma et al., 1991b; Reichert et al., 1991; Grasso
et al., 1991;Grasso et al., 1993a,b; Reichert, 1994). Some
controversy exists concerning the role of these β-subunit
peptide sequences in α/β subunit association. Whereas
some studies indicate that none of these two peptides are
involved in FSHβ binding to the α-subunit (Santa-Coloma
and Reichert, 1991), others have found that the peptides
may cause 30–80% inhibition of subunit association (Vakharia et al., 1991), thus implicating these sequences in
subunit assembly.
A third experimental strategy employed to analyse specific structural determinants for FSH function is the site-directed mutagenesis approach. In this technique, a particular
amino acid residue located within a peptide region known
to be involved in hormone function is replaced by another
residue with similar physicochemical properties. Thus, the
procedure provides a fine-tuning system to explore the
specific functions of the mutated residue. The α-subunit
has a highly active zone between amino acids 33 and 58;
this region forms a long loop with structural components
for α/β heterodimerization and human FSH binding (Liu
and Dias, 1996; Dias, 1996) (Figure 5A). In addition, the
subunit also has a site of N-linked glycosylation within this
region (Asn52), which is apparently critical for receptor
activation but not for receptor binding (Bishop et al., 1994;
271
Flack et al., 1994b; Valove et al., 1994; see below). In
HCG, the region encompassed by amino acids α36–40 is a
key determinant for initiating and maintaining α/β assembly (Bielinska et al., 1992; Xia et al., 1994). Site-directed
mutagenesis of the human common α-subunit has revealed
that Ala36 and Pro38 are involved in heterodimer formation
of CG and FSH, whereas Phe33 and Arg35 appear uniquely
important for heterodimer formation in FSH but not in
HCG (Grossmann et al., 1996). On the other hand, although HCG mutated individually in Phe33 and Arg35 of its
α-subunit failed to bind the human LH/CG receptor, these
mutations as well as mutations in the αArg42-Ser-Lys44
sequence had no effects in the association of human FSH to
its cognate receptor (Liu et al., 1993). These observations
suggest that the dimer-assembling and receptor-binding
regions in the α-subunit are not common for all glycoprotein hormones. Mutations on the sequences α55–57 and
α37–41 in human FSH significantly altered heterodimer
formation, whereas replacement of α49–51 profoundly affected receptor binding activity but not subunit association
(Grossman et al., 1996; Liu et al., 1996). Figure 5 shows a
representation of the long loop of the human α-subunit and
the HCG β-subunit loop that forms the so-called ‘seat belt’
region (HCGβ90–110); in HCG, the region comprised by
βCys90-Cys110 is wrapped over the α-subunit while remaining covalently bonded to the β-subunit through disulphide linkages (Lapthorn et al., 1994). As shown, both
sequences α55–57 and α37–41 are closely associated with
the inner surface of the seat belt, which supports the possibility that these regions in αFSH may be essential for subunit association.
The role of the COOH-terminal region of human FSHα
in receptor binding and signal transduction has been analysed in detail (Yoo et al., 1993; Zeng et al., 1995; Dias,
1996). Truncation of the short sequences αLys91-Ser92 or
αHis90-Lys-Ser92 abolished induction of cAMP production by either human FSH or HCG and reduced receptor
binding of human FSH but not of HCG (Yoo et al., 1993).
Specific substitutions, either in αTyr88, αTyr89 or αLys91
as well as in αHis90 of human FSH indicated that these
amino acids play a critical role in high affinity binding
and/or signal transduction (Zeng et al., 1995; Dias, 1996).
A 21 amino acid sequence between residues Tyr33 and
Phe53 of human FSHβ contains residues involved in α/β
subunit association as well as in ligand–receptor interaction (Santa-Coloma et al., 1990a; Roth and Dias, 1995).
The flanking amino acids of this region are apparently
important for assembly of the human FSH heterodimer.
Mutation of residues β34–37 or β48–52 yielded an FSHβ-subunit immunologically indistinguishable from the
wild type but unable to form α/β-dimers (Roth et al.,
272
A.Ulloa-Aguirre and C.Timossi
Figure 5. (A) Representation of the long loop in the human α-subunit (residues 33–58) and the human chorionic gonadotrophin
(HCG)-β90–110 seat belt region. Residues in α33–58 involved in subunit association are coloured black (Tyr37-Pro-Thr-Pro-Leu41 and
Ser55-Glu-Ser57). (B) Follicle stimulating hormone (FSH) receptor-binding site modelled using the HCG crystal structure coordinates. Unconserved residues in the HCGβ90–110 region were replaced by corresponding residues from FSHβ aligned according to cysteines. Residues in the
α-subunit long loop 33–58 affecting receptor binding are coloured black. [Reproduced from Liu and Dias (1996), with permission.]
1993). Further, a single mutation in Thr52 was sufficient to
reduce expression of heterodimeric FSH, whereas Leu38,
Val39, Gln48 and Thr50 mutants were sensitive to dissociation at low pH values, suggesting that these latter residues
may also be involved in subunit association (Roth and
Dias, 1996). On the other hand, substitution of amino acids
β37–39 caused a 20-fold reduction in receptor binding of
FSH as compared with the wild type analogue, whereas
mutations in the β34–37 and β44–47 sequences also
caused reduction in receptor binding although to a lesser
extent than the β37–39 mutant (Roth and Dias, 1995). In
addition, these studies showed that a specific mutation in
βGln48 profoundly affected receptor binding and that all of
these mutant heterodimers were still able to activate human
FSH receptor (Roth and Dias, 1995, 1996). Specific substitution of βArg35 by Lys or Asp decreased FSH binding
capacity to 71% and 98% respectively; however, the signal
transduction capacity of the mutants was unaffected when
tested at equivalent doses of FSH-binding activity (Valove
et al., 1994). Although these data suggest that receptor
binding and signal transduction may be dissociable functions involving different sites on the human FSH molecule,
it has been reported that another region in FSHβ, the
COOH-terminal region, is involved in both functional features of the FSH dimer (Lindau-Shepard et al., 1994). In
this latter study, mutation of βArg97-Gly-Leu99 to alanine
residues showed a 5-fold reduction binding activity and
replacement of the Asp93-Cys-Thr-Val96 sequence by AlaCys-Ala-Ala yielded a mutant molecule exhibiting only
residual binding potential; in addition, the capacity of both
mutants to activate the receptor and induce signal transduction was severely compromised (Lindau-Shepard et al.,
1994). Finally, a negative-specificity determinant comprised of residues 88–91 of human FSHβ (a sequence located close to the glycosylation site at αAsn52) has been
described; replacement of these residues with the corresponding HCGβ sequence (residues 94–97) allowed
human FSH to bind the LH/HCG receptor (Dias et al.,
1994; Moyle et al., 1994). In this regard, the oligosaccharide residue at αAsn52 would apparently affect the receptor
binding interface by promoting a hormone-receptor complex with enhanced signal transduction at the expense of
some energy binding (see below) (Sairam and Bhargavi,
1985).
Figure 6 summarizes the α- and β-subunit domains of
human FSH to which specific functions have been ascribed
by any of the experimental approaches described above. As
shown in Figures 5B and 6, human FSH receptor-binding
determinants are present in both subunits assembled
through subunit associations.
Role of carbohydrates
It is well known that the oligosaccharides incorporated into
the glycoprotein hormones are important for some unique
aspects of hormone function. In addition to playing an
important role in folding, subunit assembly and secretion
of the molecule (see above), they critically determine the
FSH and FSH receptor
273
Figure 6. Schematic diagram showing the domains of the α-(A) and β-(B) subunits of human follicle stimulating hormone (numbered bars) to
which specific functions have been ascribed as disclosed by immunological (Mabs), synthetic peptides (SP) and site-directed mutagenesis
(S-DM) approaches. All white horizontal bars and amino acid residues below each main numbered bar mark the residues or domains involved in
subunit association, whereas horizontal bars or squares above the numbered bars indicate the domains or resides involved in receptor-binding
activity (hatched pattern), receptor-binding/signal transduction (black pattern) or signal transduction (white pattern).
metabolic fate in circulation as well as the interaction of the
hormone with its cognate receptor (Sairam, 1989; UlloaAguirre et al., 1995).
There is compelling evidence that the sialic acid and the
sulphate residues present in the oligosaccharides of the
gonadotrophin hormones determine the metabolic clearance rate and the in vivo potency of the molecule (Morell et
al., 1971; Wide, 1986; Wide and Hobson, 1986; Fiete et al.,
1991). Terminal sulphation favours the removal of the gonadotrophin from the circulation by the action of an hepatic
reticuloendothelial cell binding protein for sulphated glycoproteins (Fiete et al., 1991); in contrast, sialic acid decreases hepatic uptake and consequently the degradation of
the sialylated molecule (Morell et al., 1971). Recent
studies employing recombinant human FSH variants lacking terminal sialic acid residues have confirmed the results
of earlier studies in which deglycosylation of the gonadotrophin was performed employing chemical or enzymatic
methods (Vaitukaitis and Ross, 1971; Yang and Papkoff,
1973); sialic acid-deficient FSH variants as well as FSH
molecules bearing only Asn-linked (GlcNAc)2-(mannose)5 oligosaccharides were rapidly cleared from the cir-
culation of rat and were also practically inactive in vivo as
compared with the recombinant wild-type variant (Galway
et al., 1990). More recently, Bishop and colleagues (1995)
have shown that both β-subunit carbohydrate residues of
FSH play a major role in determining the metabolic clearence rate and in-vivo potency of the gonadotrophin. Using
site-specifically deglycosylated recombinant FSH variants, these investigators observed that mutant molecules
bearing disrupted glycosylation consensus sequences in
either one or two of the glycosylation sites of the β-subunit
or in both subunits, exhibited a significantly higher plasma
disappearance rates and lower in-vivo biological potencies
than α-subunit deglycosylated variants, whose metabolic
clearance rate and in-vivo biopotency were slightly lower
or similar to those exhibited by the fully glycosylated variant (Bishop et al., 1995). Thus, it seems that the behaviour
of FSH in vivo is mainly determined by the ability of the
carbohydrates linked to its β-subunit [which are predicted
to be located on the periphery of the molecule (Lapthorn et
al., 1994)] to prevent or delay renal and/or hepatic clearance of the gonadotrophin.
274
A.Ulloa-Aguirre and C.Timossi
Studies employing partially or completely deglycosylated FSH variants by chemical procedures or specific
deletion of sites for N-linked glycosylation, have allowed
the determination of the functional role of the oligosaccharide residues in receptor binding and signal transduction (Manjunath et al., 1982; Sairam and Bhargavi, 1985;
Calvo et al., 1986; Sairam, 1989). Early studies using the
hydrogen fluoride procedure (which removes 70–90% of
the total carbohydrate without affecting the polypeptide
backbone of the glycoprotein) to unselectively deglycosylate the FSH molecule or one of its subunits, showed that
the carbohydrate residues linked to the α-subunit play a
dominant role in the capacity of the hormone to activate the
receptor and induce signal transduction (Sairam and Bhargavi, 1985). In these studies, binding of ovine or human
FSH to the receptor was significantly increased or remained unaltered following HF deglycosylation as disclosed by heterologous receptor-binding assays, whereas
signal transduction, measured by the ability of these variants to induce cAMP accumulation, was almost completely
abolished. Further, deglycosylated α-subunit recombined
with the intact β-subunit of ovine FSH retained its binding
ability but was inactive in inducing adenylate cyclase activation (Sairam and Bhargavi, 1985; Sairam, 1989).
More recently, site-directed mutagenesis of amino acid
residues involved in N-linked glycosylation has been used
to selectively inhibit glycosylation of the human FSH molecule (Bishop et al., 1994; Flack et al., 1994b; Valove et al.,
1994). All these studies have corroborated the crucial role
of the oligosaccharides attached to the α-subunit in signal
transduction, with particular emphasis on the carbohydrates linked to Asn52 on the α-subunit. Prevention of glycosylation at this particular site (αAsn52) resulted in either
increased (Flack et al., 1994b; Valove et al., 1994) or unaltered (Bishop et al., 1994) binding capacity as well as in a
significantly reduced signal transduction ability of the mutant molecule as assessed by either heterologous or homologous assay systems. Similar results were found when
glycosylation at both αAsn52 and αAsn78 was prevented
(Bishop et al., 1994; Flack et al., 1994). Data on the role of
the carbohydrates linked to the β-subunit are rather contradictory; in one study (Flack et al., 1994b) deletion of oligosaccharide at position βAsn7 resulted in a 2.5-fold increase
in receptor binding activity and a 2-fold decrease in its
signal-transducing potency whereas in the study from
Bishop et al. (1994) the activity of the variant was unaffected by the site-specific deletion. Further studies employing homologous assay systems, confirmed that in fact
the oligosaccharide chains of the β-subunit play a minor
role, if any, in human FSH receptor binding and signal
transduction and that the oligosaccaride at position αAsn52
is essential to trigger a biological response at the target cell
level (Valove et al., 1994). The crystallographic structure
of HCG indicates that the αAsn52 oligosaccharide is located near the central cysteine knot region of the hormone,
which is putatively involved in hormone–receptor interaction, wheras the remaining oligosaccharides are projected
toward the periphery of the molecule, away from this central core (Lapthorn et al., 1994). The finding that the oligosaccharide attached to αAsn52 of HCG is closely involved
in signal transduction (Matzuk and Boime, 1989) strongly
suggests that the carbohydrate-dependent structural requirements which determine receptor activation in both
gonadotrophins may be similar.
Sialic acid residues and to a lesser extent sulphate residues play a key role in determining the charge isoform
distribution of FSH (Baenziger and Green, 1988; UlloaAguirre et al., 1992, 1995). Multiple charge FSH isoforms
have been isolated from anterior pituitary extracts, serum
and urine of several animal species, including man (UlloaAguirre et al., 1988a; Chappel, 1995; Ulloa-Aguirre et al.,
1995). Recently, recombinant human FSH has been obtained by expressing the human FSHα- and β-subunits
complementary DNAs in Chinese hamster ovary cells
(CHO cells) (Keene et al., 1989; Cerpa-Poljak, 1993) and
human embryonic kidney cell lines (HEK-293) (Flack,
1994a). However, in contrast to recombinant FSH produced by HEK-293 cells, FSH preparations obtained from
CHO cells do not contain the full spectrum of human FSH
isoforms found in the pituitary or in circulation (Flack et
al., 1994a; Ulloa-Aguirre et al., 1997). Further, recombinant FSH expressed in CHO cells contains no bisecting
GlnNAc residues (structures c and g in Figure 2), exhibits
fewer complex oligosaccharides and bears an increased
proportion of simple carbohydrate structures as compared
to its counterparts isolated from the pituitary and the urine
of postmenopausal women (Hard et al., 1990; Harris et al.,
1996). Separation of human anterior pituitary FSH by
charge-based procedures has disclosed the existence of 20
or more charge isoforms of the hormone (Stanton et al.,
1996; Zambrano et al., 1996). As expected, the more
acidic/sialylated variants exhibit the longest plasma halflife and the highest in-vivo bioactivity when assessed by
the ovarian weight augmentation test in immature mice
(Ulloa-Aguirre et al., 1992a; Wide, 1986; Wide and Hobson, 1986). On the contrary, it has been shown that less
acidic isoforms may exhibit higher receptor binding activity and in-vitro biological potency than the more acidic
counterparts when such functional parameters are expressed as the receptor-binding activity/immunoactivity or
the bioactivity/immunoactivity FSH ratios, respectively
(Ulloa-Aguirre et al., 1995; Zambrano et al., 1996). Al-
FSH and FSH receptor
though the use of these relationships to express the potency
of the hormone at the target cell level has raised some
controversy due to the likelihood that gonadotrophin heterogeneity may alter immunoreactivities (Chappel, 1990;
Simoni et al., 1994; Stanton et al., 1996; Robertson et al.,
1997), it has been shown that the radioreceptor activity and
in-vitro bioactivity of highly purified charge isoforms may
also vary over a 5–8-fold range when expressed on the
basis of defined protein content (Stanton et al., 1996).
These studies strongly suggest a role for terminal sialic acid
residues in determining the interaction of the heterogeneous FSH variants with its receptor. However, the origin of
FSH heterogeneity cannot be attributed fully to the variability in negatively charged terminal carbohydrate residues (sialic acid and/or sulphate residues) since a large
number of different human FSH glycoforms, exhibiting
particular receptor-binding activity and in-vitro bioactivity, may be synthesized, and many molecular isomers may
have the same charge yet different oligosaccharide composition as disclosed by lectin binding studies (UlloaAguirre et al., 1988b, 1992b). When isoforms of pituitary
FSH identified by charge-based procedures are examined
for their ability to bind to Concanavalin A, the less acidic
isoforms exhibit an increased affinity for the lectin, thus
indicating the presence of greater proportions of high mannose and/or hybrid structures in these isoforms (UlloaAguirre et al., 1988b, 1992b; Creus et al., 1996).
Furthermore, it has been found that some of these particular naturally occurrring underglycosylated variants may
behave as FSH antagonists in vitro (Dahl et al., 1988; Timossi et al., 1998) and that their inhibitory properties are
exerted at or independently of downstream steps of cAMP
formation (Timossi et al., 1998). The mechanisms subserving the pleiotropic actions of the human FSH glycoforms
are not clearly understood and several possible mechanisms have been suggested (Ulloa-Aguirre et al., 1995; Arey
et al., 1997; Timossi et al., 1998). In this regard, Arey et al.
(1997) have proposed an interesting mechanism through
which the gonad may respond in diverse ways to complex
hormonal signals such as those presented by certain circulating FSH glycoforms. These investigators observed that
underglycosylated insect cell-expressed recombinant FSH
binds distinctly to the FSH-R allowing the ligand–receptor
complex to acquire diverse conformations that may trigger
either stimulatory or inhibitory responses depending on the
level of receptor-transducer activation. Apparently, this
dual effect of the underglycosylated variant may be exerted
via promiscuous receptor–G protein coupling in which
several signal transducer molecules (e.g. Gs and Gi/o proteins) may become activated depending on the dose of the
275
ligand and the particular ligand glycosylation-dependent
changes in receptor conformation.
It has been consistently found that the distribution of the
intrapituitary and secreted FSH glycoforms changes during specific physiological conditions such as puberty
(Chappel et al., 1983b; Chappel and Ramaley, 1985; UlloaAguirre et al., 1986; Ulloa-Aguirre et al., 1990; Phillips et
al., 1997) and the ovarian cycle (Padmanabhan et al., 1988;
Wide and Bakos, 1993; Zambrano et al.,1995), thus indicating that FSH pleomorphism is a hormonally regulated
phenomenon. In terms of endocrine regulation of FSH glycoforms, there is growing evidence indicating that hypothalamic and gonadal inputs (e.g. GnRH and oestrogens)
are involved not only in regulating synthesis and secretion
of FSH but also in modulating the molecular nature of the
hormone and therefore its biological attributes (Wide and
Naessén, 1994; Ulloa-Aguirre et al., 1995; Wide et al.,
1995). In this regard, it has been reported that the activity of
some glycosyltransferases (e.g. sialyltransferases and GalNAc transferase) and mannosidases may be modulated at
the pituitary level by thyroid hormones (Helton and
Manger, 1994) and oestrogens (Dharmesh and Baenziger,
1993; Damián-Matsumura et al., 1998), thereby affecting
sialylation and sulphation of all pituitary glycoprotein hormones. Although these observations concurrently suggest
that the phenomenon of FSH pleomorphism may be functionally relevant, more studies are necessary before assigning physiological and clinical importance to FSH
heterogeneity.
Conclusions
From the previous discussion, it is clear that multiple structural determinants are involved in FSH and FSH-R function and that despite their structural complexity both
molecules exhibit a high degree of plasticity and diversity
that allows formation of distinct ligand–receptor complexes capable of selectively activating or deactivating a
variety of signalling pathways. Knowledge and mapping of
these structural determinants and functional epitopes for
intracellular and extracellular hormone action as well as
molecular characterization of those factors that may potentially interfere in hormone action (Fauser, 1996) are of
paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action
and FSH-R function but also for the rational design of
analogues with predicted properties and effects. For
example, based on the findings that multiple site interactions between FSH and its receptor exist, there is the potential for using synthetic linear sequences of the ligand to
276
A.Ulloa-Aguirre and C.Timossi
obtain selective agonistic or antagonistic effects. In this
regard, it has been recently shown that a synthetic peptide
amide corresponding to residues 34–37 of human FSHβ
may function as an FSH antagonist in vitro and when administered at high doses it is capable either of disrupting the
normal mouse oestrous cycle or of accelerating the onset of
puberty in both male and female mice (Grasso and Reichert, 1996; Grasso et al., 1997). On the other hand, application of recombinant technology has allowed the
engineering of a variety of analogues with distinct biological features and therapeutic potential. For example, the use
of site-directed mutagenesis and gene transfer techniques
to add the carboxyl-terminal peptide extension of the
HCGβ-subunit to the FSHβ-subunit has resulted in an FSH
agonist with receptor-binding and steroidogenic activities
indistinguishable from those of the wild-type FSH, but
possessing a markedly increased circulating half-life and
in-vivo potency (Fares et al., 1992; Lapolt et al., 1992).
Such a ‘hybrid’ FSH compound may be potentially useful
to treat male subjects with hypogonadotrophic hypogonadism, in whom daily administration of FSH for a long period
of time is often necessary to achieve reproductive competence. Likewise, it is currently feasible to convert heterodimeric gonadotrophins to genetically linked single chains
with enhanced in-vivo potency and in-vitro stability, which
also may obviate the need of frequent administration of the
heterodimeric molecule in infertility patients (Sugahara et
al., 1996). The fact that the carbohydrate residues present
in gonadotrophins play a key role in receptor activation and
signal transduction, also offers interesting alternative
means for analogue design. In fact, it has been extensively
demonstrated that both deglycosylated and underglycosylated human gonadotrophin variants may behave as antagonists of the wild-type molecules in in-vitro systems
(Sairam and Manjunath, 1982, 1983; Sairam, 1989; Ji and
Ji, 1990; Dunkel et al., 1993; Keene et al., 1994; Arey et
al., 1997; Timossi et al., 1998). Whether these gonadotrophin variants may also act as antagonists in vivo still remains to be investigated. Finally, the possibility of
developing drugs that might exert inhibition at the level of
receptor/G protein interaction rather than at the level of
ligand–receptor binding cannot be overlooked, particularly
considering recent studies showing that different receptor
domain peptides may inhibit receptor-mediated signalling
in a variety of G-protein cell receptors, including the FSHR (Hawes et al., 1994; Grasso et al., 1995a,b,c; Quian et
al., 1998; Ulloa-Aguirre et al., 1998). The development of
this array of analogues will undoubtedly expand the therapeutic horizons in reproductive medicine for the coming
century.
Acknowledgements
Studies performed in the author’s laboratory have been supported
by grants from the Consejo Nacional de Ciencia y Tecnología
(CONACyT, Mexico), the Programa Latinoamericano de Capacitación e Investigación en Reproducción Humana (PLACIRH, México) and the Rockefeller Foundation (New York,
USA). The authors are deeply indebted to Dr Scott C.Chappel,
Ares Advanced Technology, Randolph, Massachusetts and for
his helpful suggestions in the preparation of this manuscript. We
also thank Dr Deborah Alemán-Hoey, University of Vermont,
Burlington, VT, USA, for language editing.
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