REVIEW ARTICLE published: 26 February 2015 doi: 10.3389/fendo.2015.00026 Structure–function relationships of glycoprotein hormones and their subunits’ ancestors Claire Cahoreau, Danièle Klett andYves Combarnous* Physiologie de la Reproduction et des Comportements (PRC), Centre National de la Recherche Scientifique, INRA, Nouzilly, France Edited by: Glycoprotein hormones (GPHs) are the most complex molecules with hormonal activity. Hubert Vaudry, University of Rouen, They exist only in vertebrates but the genes encoding their subunits’ ancestors are found France in most vertebrate and invertebrate species although their roles are still unknown. In the Reviewed by: Francisco Gaytán, University of present report, we review the available structural and functional data concerning GPHs and Cordoba, Spain their subunits’ ancestors. Bruno Querat, Université Keywords: glycoprotein hormones, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating Paris-Diderot, France hormone, evolution, molecular, structure–activity relationship *Correspondence: Yves Combarnous, Physiologie de la Reproduction et des Comportements (PRC), Centre National de la Recherche Scientifique, INRA, Nouzilly 37380, France e-mail: yves.combarnous@ tours.inra.fr INTRODUCTION In the present paper, we will consider the structure–function Glycoprotein hormones (GPHs) are the most complex mole- relationships of GPHs and of their receptors (GPHRs) to better cules with hormonal activity. They include three pituitary hor- understand their interactions and the subsequent steps in their mones, the gonadotropins follicle-stimulating hormone (FSH; target cells stimulation. follitropin) and luteinizing hormone (LH; lutropin) as well as thyroid-stimulating hormone (TSH; thyrotropin) (1). Only in pri- STRUCTURE OF GLYCOPROTEIN HORMONES AND THEIR mates (2) and equidaes (3), a chorionic gonadotropin (CG) is also ANCESTORS secreted by the placenta. Since 1971, GPHs are known to consist of two different glycopro- The GPHs exist only in vertebrates and appeared during evolu- tein subunits, called a and b, that are non-covalently associated tion along with the pituitary. Nevertheless, genes coding for mol- (9–12). This heterodimeric structure has been known for a long ecules related to GPHs subunits were identified in all vertebrates time to be mandatory for their respective biological functions. studied and in most invertebrates (4–6). The saccharide part in GPHs represents as much as 20–45% of The gonadotropins FSH and LH play a central role in ver- their total mass (11,13,14) and has been shown to be indispensable tebrate reproductive function (7, 8) as they convey the inte- for their in vivo bioactivity (15, 16). It is therefore important to get grated central information from the hypothalamic–pituitary com- as much information as possible concerning both their polypep- plex toward gonads in both males and females. Indeed, inter- tide and polysaccharide portions as to decipher their respective nal (mainly endocrine) and external (photoperiod, congeners) roles. information are integrated at the hypothalamus level by pul- More recently, genes encoding for proteins related to the GPH satile gonadotropin-releasing hormone (GnRH) secretion. In a and b subunits were found in both vertebrates and invertebrates mammals, GnRH is released by GnRH neurons in the portal and were named GPA2 and GPB5, respectively (4, 6, 17) and are hypothalamic–pituitary system through which it enters into the considered as the molecular ancestors of GPH subunits (Figure 1). anterior pituitary. In fishes, GnRH neurons release GnRH directly Recombinant GPA2 and GPB5 have been produced using plasmids into the pituitary. In all cases, GnRH stimulates the secretion encompassing the coding regions from these genes. These recom- of both gonadotropins FSH and LH by the anterior pituitary binant molecules were characterized using various immunoassays but their secretions are also differentially modulated by gonadal and in vitro bioassays. The natural GPA2 or GPB5 proteins have feed-backs through the action of steroid hormones and protein never been isolated but were detected in adult rat pituitaries by factors. immunohistology and western blotting using antibodies raised The TSH is also secreted by the antehypophysis but under the against the recombinant proteins (6). The putative GPA2/GPB5 control of the hypothalamic neuropeptide thyrotropin releasing heterodimer has been described to exert thyrostimulating activity hormone (TRH) and is modulated by thyroid feed-back through (i.e., the name thyrostimulin coined for it) (6). the action of thyroxin (T4) or tri-iodo-thyronine (T3). The placental gonadotropins (hCG in human; eCG in the mare) POLYPEPTIDE PART are secreted by trophoblast cells under no known control by any The GPHs a- and b-subunits are encoded by different releasing hormone. genes (i.e., they do not originate from post-translational www.frontiersin.org February 2015 | Volume 6 | Article 26 | 1 Cahoreau et al. Structure-function of glycoprotein hormones FIGURE 1 | Glycoprotein hormones’ emergence and evolution. The origin of vertebrates (18). Both GPA2 and GPB5 are cystine-knot GPH a- and b-subunits genes derived from GPA2 and GPB5 genes, proteins with three loops and might derive from the same ancestral respectively after the two rounds of full genome duplication (2R) at the molecule. proteolytic maturation of a unique precursor like insulin two or three along the sequence leaving three sequence portions subunits). without cysteines that are expected to form loops. The a-subunits of all GPHs in a given species are encoded by a In the a-subunits, two potential N-glycosylation amino-acid same and unique gene that is expressed in pituitary gonadotrope sequences (Asn–X–Ser/Thr) are found and both of them are and thyrotroph cells of all vertebrates as well as in chorionic syn- indeed occupied by oligosaccharide chains. cytiotrophoblastic cells of primates and equidaes. Therefore, the The amino-acid sequences of common a-subunits from all GPHs a-subunits all exhibit the same amino-acid sequence in a vertebrates exhibit a fairly high percentage of identity. This is given species. well illustrated by the fact that it is possible to recombine these By contrast, the b-subunits are different and specific for each a-subunits with b-subunits from the other GPHs and from hormone. Therefore, there are at least three genes encoding b- phylogenetically distant species (20, 21). subunits in all vertebrate species, namely FSHb, LHb, and TSHb. The GPA2 amino-acid sequences derived from the gene In the human species, there are not only one but several genes sequences in numerous species indicate that there are two poten- encoding the hCGb subunit. tial N-glycosylation sites but one is not at the same location as in The two subunits are thus co-translated and they non- a-subunits. covalently combine in the endoplasmic reticulum of gonadotrope The specific b-subunits (FSHb, LHb, TSHb, and CGb) (FSH, LH), thyrotroph (TSH), or trophoblast (CG) cells. No infor- polypeptide sequences exhibit around 105–150 amino-acids (19). mation is available for the time being concerning the natural GPA2 Although different, the b-subunit amino-acid sequences exhibit and GPB5 proteins. large similarities which are probably largely due to their main common characteristic which is to associate with a common Primary structure a-subunit. The conserved sequences in the b-subunits are impor- All GPH subunits sequences as well as those of GPA2 and GPB5 tant for (1) direct interaction with a and (2) similar global exhibit a signal peptide at their N-terminus indicating that all these folding. molecules are secreted glycoproteins. All b-subunits possess 12 cysteine residues which are all impli- The common a-subunits (GPA1) of mammalian GPHs after cated in the six intra-chain disulfide bridges. In spite of their excision of their signal peptide exhibit 92 or 96 amino-acid different sequences that determine the specificity of the different sequences among which 10 are cysteine residues. Since no cysteine hormones, the b-subunits share a number of common features. residue is in the reduced state, the a-subunits possess five disulfide In particular, the positions of the 12 cysteine residues in their bridges (Figure 2). In other species, the maturated a subunits also sequences are highly conserved so that they are expected to share count approximately 90–100 amino-acid residues (19). a common global folding (see below). The repartition of cys- The amino-acid sequences of a-subunits are very well con- teines along the amino-acid sequences of b-subunits also leaves served among vertebrate species and in particular the positions sequences without cysteines that are expected to form three loops of the 10 cysteine residues. The residues are found in groups of (L1, L2, L3). Frontiers in Endocrinology | Neuroendocrine Science February 2015 | Volume 6 | Article 26 | 2 Cahoreau et al. Structure-function of glycoprotein hormones FIGURE 2 | Glycoprotein hormone a- and b-subunits’ primary structures. The relative positions of Cys residues and N- and O-saccharide chains along the amino-acid sequences are shown. Some mammalian b-subunits (hCGb, eCGb, and eLHb) pos- A possible explanation for the difference in b-structure pro- sess an extension of approximately 30 amino-acid residues at portion as determined by CD and X-ray diffraction is that the first their C-terminus that is called carboxy-terminal peptide (CTP). one is carried out in solution whereas the second