Molecular cloning and pharmacological characterization of two novel GnRH receptors in the lamprey (Petromyzon marinus)

Abstract

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate.

Fig. 1.

Alignment of human, mouse, African green monkey, bullfrog, leopard gecko, chicken, zebrafish, and lamprey GnRH receptor sequences. The sequences (see Fig. 5, accession details) were aligned with ClustalW2 (28), and an image was generated by editing and annotating, the style based on the output initially produced with BOXSHADE 3.21. Ubiquitously conserved amino acid residues are shown with dark background shading, and amino acids with conserved substitutions are shown with light shading Semiconserved substitutions are shown with a dot on the consensus line. Bold horizontal lines indicate predicted TMD (TMD 1–7). GnRH receptor subtypes are labeled with ordinal numbers according to the published terminology (7, 14, 47, 50) and grouped together. Residues, microdomains, and motifs discussed in the text are bracketed above and below the alignments.

Fig. 2.

Tissue distribution of lGnRH-R-2 (A) and lGnRH-R-3 (B) mRNA expression in male and female lampreys. Arrows indicate the corresponding products. RNA integrity and cDNA production were verified by amplification of elongation factor 1α (C). The expected DNA fragment sizes were 855 and 589, respectively. Each amplified fragment of lGnRH-R-2 and -3 spans all three exons.

Fig. 3.

Ligand binding of lGnRH-R-1, lGnRH-R-2, and lGnRH-R-3. Competitive displacement of [¹²⁵H]cGnRH-II with serial dilutions (10⁻¹¹m to 10⁻⁵m) of lGnRH-I (■), (10⁻¹¹m to 10⁻⁵M) lGnRH-II (▴) and (10⁻¹¹m to 10⁻⁵m) lGnRH-III (▾) in COS-7 cells transiently transfected with lGnRH-R-1 (A), lGnRH-R-2 (B), and lGnRH-R-3 (C) expression constructs. The data presented are from four independent experiments each performed in triplicate. Nonspecific binding, determined in nontransfected cells, was subtracted from maximal counts per minute.

Fig. 4.

IP production of lGnRH-R-2 and lGnRH-R-3 in response to (10⁻¹¹ m to 10⁻⁴m) lGnRH-I (■), (10⁻¹¹m to 10⁻⁴m) lGnRH-II (▴), and (10⁻¹¹m to 10⁻⁴ m) GnRH-III (▾) in COS-7 cells transiently transfected with lGnRH-R-2 (A) and lGnRH-R-3 (B) expression constructs. The data are from two to three independent experiments performed in triplicate.

Fig. 5.

The molecular phylogenetic analysis was constructed using maximum-likelihood method using 68 deduced amino acid sequences of the vertebrate and invertebrate GnRH receptors. Numbers on the branches indicate bootstrap probabilities following 1000 replications in constructing the tree. The DDBJ/ MBL/ GenBank accession numbers of amino acid sequences used for the phylogenetic analysis are as follows: African cichlid 1, Haplochromis burtoni (AY705931.1); African cichlid, H. burtoni (AY028476.1); African green monkey, Cercopithecus aethiops (AF353988.1); amberjack, Seriola dumerili (AJ130876.1); African clawed frog 1, Xenopus laevis (AF172330.1); African clawed frog 2, X. laevis (AF257320.1); Amphioxus 1, Branchiostoma floridae (EU433377.1); Amphioxus 2*, B. floridae (EU433378.1); Amphioxus 3, B. floridae (EU433380.1); Amphioxus 4, B. floridae (FJ426561.1); brown frog 1, Rana dybowskii (AF236879.2); brown frog 2, R. dybowskii (AF236877.2); brown frog 3, R. dybowskii (AF236878.1); bullfrog 1, R. catesbeiana (AF144063.1); bullfrog 2, R. catesbeiana (AF153913.1); bullfrog 3, R. catesbeiana (AF224277.1); chicken 1, Gallus gallus (AJ304414.1); chicken 2, G. gallus (AY895154.1); cow, Bos Taurus (U00934.1); dog, Canis lupus familiaris (AF206513.1); European sea bass 1A, Dicentrarchus labrax (AJ606683.2), European sea bass 2A, D. labrax (AJ419594.1); European sea bass 2B, D. labrax (AJ606686.2); fruit fly C, Drosophila melanogaster (AE014134.5); fruit fly B, D. melanogaster (AE014134.5); fruit fly A, D. melanogaster (AE014134.5); gecko 1/IIIl, Eublepharis macularius (DQ269481.1); gecko 2, E. macularius (AB109032.1); gecko 3, E. macularius (DQ269482.1); goat, Capra hircus (EF150356.1); goldfish B, Carassius auratus (AF121846.1); goldfish A, C. auratus (AF121845.1); guniea pig, Cavia porcellus (AF426176.3); human, Homo sapiens (NM_000406.2); Japanese eel, Anguilla japonica (AB041327.1); Japanese medaka 1, Oryzais latipes (AB057677.1); Japanese medaka 2, O. latipes (AB057676.1); Japanese medaka 3, O. latipes (AB083364.1); mangrove kill fish, Kryptolebias marmoratus (DQ996268.2); marmoset 2, Callithrix jacchus (AF368286.1); mouse, Mus musculus (L01119.1); mummichog, Fundulus heteroclitus (AB426466.1); North African catfish 1, Clarias gariepinus (X97497.2); North African catfish 2, C. gariepinus (AF329894.1); Norway rat, Rattus norvegicus (NM_031038.3); octopus, Octopus vulgaris (AB185200.1); Pejerrey 1B, Odontesthes bonariensis (DQ875596.1); pig, Sus scrofa (L29342.1); pufferfish, Tetraodon nigroviridis (AB212821.1); pufferfish, T. nigroviridis (AB212816.1); pufferfish, T. nigroviridis (AB212819.1); pufferfish, T. nigroviridis (AB212825.1); rabbit, Oryctolagus cuniculus (AY781779.1); rainbow trout, Oncorhyncus mykiss (AJ272116.1); rhesus monkey 2, Mucaca mulatta (AF353987.1); rat, Rattus sp (AF353987.1); sea lamprey 1, Petromyzon marinus (AF439802.1); sea lamprey 2, P. marinus (DQ915103); sea lamprey 3, P. marinus (DQ915102); sheep, Ovis aries (X72088.1); striped bass, Morone saxatilis (AF218841.1); tunicate 1, Ciona intestinalis (AY742888.1); tunicate 2, C. intestinalis (AY742889.1); tunicate 3 C. intestinalis (AY742890.1); tunicate 4, C. intestinalis (AY742891.1); tilapia type 1, Oreochromis niloticus (AB111356.2); tilapia type 2, O. niloticus (AB111357.2); zebrafish 1, D. rerio (NM_001144980.1); zebrafish 2, Danio rerio (NM_001144979.1); zebrafish 3, D. rerio (NM_001177450.1); zebrafish 4, D. rerio (NM_001098193.1); oxytocin receptors: human oxytocin R, human oxytocin receptor (NM_000916.3) was used as the outgroup. Fruitfly adipokinetic hormone receptors were used because they are the closest insect relatives to GnRH-R. *, A second C-terminal tail truncated sequence for this GnRH receptor subtype was included in the analysis. (EU433379.1).