br Evolution of multiple GnRH forms and GnRH receptors acros

Evolution of multiple GnRH forms and GnRH receptors across vertebrates GnRH is an evolutionary ancient peptide that first appears prior to the protostome-deuterostome split, and is a member of the GnRH-adipokinetic hormone-corazonin superfamily of peptides (Roch et al., 2011, Roch et al., 2014). Excluding the hagfish, which some argue is a craniate rather than a true vertebrate, the 18 known members of the vertebrate GnRH family are mostly decapeptides, each encoded by a prohormone gene with similar exon-intron structures, and can be grouped into a number of classes based on a combination of genomic structure, synteny, distribution, and function (Roch et al., 2011, Roch et al., 2014, Table 2). Although controversial and up to five GnRH classes has been proposed, it is now commonly accepted that vertebrate GnRHs can be grouped into three classes (Decatur et al., 2013). Class 1 GnRHs (GnRH1, ≥12 isoforms) include the classical hypophysiotropic mGnRH and other hypophysiotropic GnRHs; in addition, an active post-translationally modified GnRH1, [hydroxy-Pro9]-mGnRH, has been recently described. Class 2 GnRHs (GnRH2, ≥2 isoforms) are typified by the mid-brain chicken GnRH-II (cGnRH-II) form involved in the regulation of behaviour, gonadal actions, and other functions; but, GnRH2 is not generally thought of as hypophysiotropic. Class 3 GnRHs (GnRH3, ≥4 isoforms) are represented by salmon GnRH (sGnRH) and seems to be specific to fishes, and functions as the hypophysiotropic form in teleost species where GnRH1 is lost or absent (Decatur et al., 2013, Roch et al., 2014). At least two GnRHs are present in the Clindamycin Phosphate of all vertebrate classes (commonly cGnRH-II, plus one of either GnRH1 or GnRH3), although the presence of all three GnRH types has been demonstrated in some fish species (Karigo and Oka, 2013). cGnRH-II has also been detected in the pituitary of goldfish, striped bass, zebrafish, African catfish, European eel and Senegalese sole where direct innervation of the pars distalis occurs as in other teleosts; interestingly in the goldfish and zebrafish, the origin of the pituitary cGnRH-II has been mapped to the mid-brain GnRH2 neurons (Yu et al., 1991, Chang and Jobin, 1994, Sherwood et al., 1993a, Sherwood et al., 1993b, Kim et al., 1995, Gothilf et al., 1995, Kah et al., 1986, Xia et al., 2014, Guzmán et al., 2009). In the case of the Senegalese sole, the pituitary content of GnRH2 (cGnRH-II) is actually greater than those of GnRH1 (seabream (sb)GnRH) and GnRH3 (sGnRH; Guzmán et al., 2009). In the striped bass, the contents of the three GnRHs have been measured at a ratio of 100:10:1 for sbGnRH (GnRH1):cGnRH-II (GnRH2):sGnRH (GnRH3; Gothilf et al., 1995), respectively. These observations suggest more than one endogenous GnRH form can regulate pituitary cell functions, and the mid-brain GnRH2 system may exert hypophysiotropic effects in some species. Multiple forms of GnRH receptors (GnRHRs) also exist, all belonging to the 7 transmembrane domain-containing class A superfamily of G-protein-coupled receptors (GPCRs). The first GnRHR was cloned from mouse pituitary tissues and is unusual in that it lacks the cytoplasmic C-terminal domain important for receptor internalization and desensitization, processes common to all class A GPCRs (Tsutsumi et al., 1992). Likewise, human and other mammalian GnRHRs lack the C-terminal tail, and GnRHRs lacking the intracellular tail have since been identified in lower vertebrates, including coelacanth and some cartilaginous fishes (Roch et al., 2014, Williams et al., 2014). Interestingly, GnRHRs with the typical C-terminal tail of GPCRs are found in both mammalian and nonmammalian vertebrate species. Several classification schemes for GnRHRs have been proposed with a current scheme grouping all GnRHRs lacking an intracellular tail as GnRHR Is, while all other GnRHRs possessing C-terminal tails are classified as GnRHR IIs (Roch et al., 2014, Williams et al., 2014). GnRHR IIs are subdivided into IIa and IIb, with IIb encompassing GnRHRs formerly classified as those possessing elongated tails, but are still mammalian GnRHR I-like receptors (Roch et al., 2014), while GnRHR IIa are further categorized into IIa-1, IIa-2 and IIa-3 (Williams et al., 2014). Genes encoding GnRHR I and II are thought to be present in the ancestor to all vertebrates. Interestingly, GnRHR II is absent or non-functional in typical mammalian study models systems (rat or human, respectively), but other mammalian species still express both GnRHR I and II (e.g., pigs, macaques, koalas and opossums; Busby et al., 2014, Roch et al., 2014). On the other hand, the GnRHR I gene appears to have been lost in the ray-fin fishes, amphibian, reptile and bird lineages; but multiple isoforms of one GnRHR II subtype are often found as a result of duplication of the remaining gene(s), particularly in teleost (Roch et al., 2014). GnRH binding sites in GnRHR I and II forms are highly conserved but amino acid residues in the second and fourth transmembrane domains are known to confer selectivity between GnRH1 and GnRH2 (Forfar and Lu, 2011, Coetsee et al., 2008). In general, homologous GnRH(s) bind GnRHRs from the same species better than heterologous form(s) (Roch et al., 2014); but ligand selectivity between GnRHRs to endogenous GnRH forms has also been shown. For example, the two cloned goldfish GnRHRs, named goldfish GfA and GfB, and belonging to GnRHR IIb group, both bind cGnRH-II (GnRH2) with greater affinity than sGnRH (GnRH3) in expression systems (Illing et al., 1999). Likewise, two chicken GnRHRs (originally named cGnRH-R-I and cGnRH-R-III, both belonging to the GnRHR II group) are selective for cGnRH-II (GnRH2) relative to cGnRH-I (GnRH1) in expression systems (Joseph et al., 2009).