Dynamic evolution of the GnRH receptor gene family in vertebrates

Abstract

Background: Elucidating the mechanisms underlying coevolution of ligands and receptors is an important challenge in molecular evolutionary biology. Peptide hormones and their receptors are excellent models for such efforts, given the relative ease of examining evolutionary changes in genes encoding for both molecules. Most vertebrates possess multiple genes for both the decapeptide gonadotropin releasing hormone (GnRH) and for the GnRH receptor. The evolutionary history of the receptor family, including ancestral copy number and timing of duplications and deletions, has been the subject of controversy.

Results: We report here for the first time sequences of three distinct GnRH receptor genes in salamanders (axolotls, Ambystoma mexicanum), which are orthologous to three GnRH receptors from ranid frogs. To understand the origin of these genes within the larger evolutionary context of the gene family, we performed phylogenetic analyses and probabilistic protein homology searches of GnRH receptor genes in vertebrates and their near relatives. Our analyses revealed four points that alter previous views about the evolution of the GnRH receptor gene family. First, the "mammalian" pituitary type GnRH receptor, which is the sole GnRH receptor in humans and previously presumed to be highly derived because it lacks the cytoplasmic C-terminal domain typical of most G-protein coupled receptors, is actually an ancient gene that originated in the common ancestor of jawed vertebrates (Gnathostomata). Second, unlike previous studies, we classify vertebrate GnRH receptors into five subfamilies. Third, the order of subfamily origins is the inverse of previous proposed models. Fourth, the number of GnRH receptor genes has been dynamic in vertebrates and their ancestors, with multiple duplications and losses.

Conclusion: Our results provide a novel evolutionary framework for generating hypotheses concerning the functional importance of structural characteristics of vertebrate GnRH receptors. We show that five subfamilies of vertebrate GnRH receptors evolved early in the vertebrate phylogeny, followed by several independent instances of gene loss. Chief among cases of gene loss are humans, best described as degenerate with respect to GnRH receptors because we retain only a single, ancient gene.

Figure 1

cDNA and deduced amino acid sequence of the GnRH receptor IIa-2 gene in axolotls. Within the cDNA sequences, lowercase letters indicate untranslated regions and uppercase letters indicate the open reading frame; underlining indicates the polyadenylation site. Transmembrane domains were predicted using the HMMTOP 2.0 server [30] and are indicated above the relevant portion of the sequence. Putative G-protein interaction sites are inferred based on homology with Xenopus laevis [23] and are indicated with a black background. Transparent boxes indicate putative phosphorylation sites (>80% probability) predicted using the NetPhosK server [31].

Figure 2

cDNA and deduced amino acid sequence of the GnRH receptor IIa-3 gene in axolotls. Analysis and formatting as described in Figure 1.

Figure 3

cDNA and deduced amino acid sequence of the GnRH receptor IIb gene in axolotls. Analysis and formatting as described in Figure 1.

Figure 4

Comparison of deduced amino acid sequences for the three amphibian GnRH receptor genes, aligned using MUSCLE [ 32 ] . Each of the three genes identified in frogs has a clear ortholog in axolotls. Amino acids that are highly conserved (>80% sequence identity) are indicated with the darkest shading; lighter shading indicates 60-80% identity; the lightest shading indicates 40-60% identity; and a white background indicates low conservation (<40% identity). Latin names of species and GenBank accession numbers for sequences are provided in Additional file 1: Table S1.

Figure 5

Phylogram from Bayesian analyses depicting the evolutionary relationships among the genes encoding receptors for GnRH and other peptides. Two amphioxus sequences, A and B, differ by a single amino acid; thus, only one was included in the analysis. Colored backgrounds emphasize strongly-supported, monophyletic subfamilies of GnRH receptors. Symbols indicate three categories of support value: triangles, posterior probability of 0.90-0.95; squares, posterior probability of 0.95-0.99; circles, posterior probability of 1.0. ACPR = adipokinetic hormone/corazonin-related peptide receptor; AKHR = adipokinetic hormone receptor; CRZR = corazonin receptor; OTR = oxytocin receptor; V1bR = type 1b vasopressin receptor. The scale bar depicts a branch length corresponding to 0.3 amino acid substitutions per site. Latin names of species and accession numbers for sequences are provided in Additional file 1: Table S1.

Figure 6

Complete open reading frame sequences for Type I GnRH receptors, aligned using MUSCLE [ 32 ] . The Type I receptors identified in chimaeras and coelacanths (uppermost sequences) share high sequence similarity with those from mammals as well as identical intron-exon boundaries (arrowheads) and the lack of a cytoplasmic C-terminal domain. Amino acids that are highly conserved (>80% sequence identity) are indicated with the darkest shading; lighter shading indicates 60-80% identity; the lightest shading indicates 40-60% identity; and a white background indicates low conservation (<40% identity). Transmembrane domains were predicted using HMMTOP 2.0 [30] and are indicated with dark lines surrounding the relevant portion of the sequences. Latin names of species and accession numbers for sequences are provided in Additional file 1: Table S1.

Figure 7

Results of constrained topology tests using a stepping-stone model for Bayes Factor analyses [ 37 ] . The shape of each symbol indicates the type of topology test used: plus inside a circle indicates a positive constraint, and slash inside a circle indicates a negative constraint. The color of each symbol indicates whether the constrained topology was significantly worse (red), better (green), or not significantly different from (black) the null hypothesis. More than one test is symbolized on each tree and constraints were applied only to the branch indicated; ancestral and derived branches were not constrained. Monophyletic clades from Figure 5 are summarized as triangles, but an individual sequence for the chimaera Type I receptor is listed separately in some cases. Positive constraints (circled plus) forced monophyly for the labeled branch and negative constraints (circled diagonal line) prohibited monophyly for the labeled branch. Arrows at the base of trees are symbols that summarize all ancestral clades.

Figure 8

Logos for representative HMM profiles from each of the five subfamilies of GnRH receptors. The physical location of the region used to construct this HMM model, named ‘TM4 to 5′ in Additional file 3: Table S3 roughly corresponds with the C-terminal half of trans-membrane domain 4 to the C-terminus of transmembrane domain 5 (Figure 10). The relative entropy score for each position in the HMM profile is indicated by the height of stacked single-letter abbreviations for amino acids. Vertical red lines indicate the presence of an indel and pink shading to the right of the line represents an estimate of size variation for each respective indel. Logos were generated using LogoMat-M [50].

Figure 9

Summary of the hypothesized evolutionary history of the GnRH receptor family in vertebrates. Boxes and their respective colors represent major groups of paralogous gene family members, as illustrated in Figure 5. Filled boxes symbolize gene presence and open boxes symbolize gene absence. “-” in an open box indicates the initial loss of a gene and “+” indicates the initial gain of a gene. “?” indicates that insufficient data are available to determine whether the ancestral condition is presence of one, two, or three copies of a Type IIa-3 receptor, and “*” indicates receptors that arose either through duplication or divergence. “WGD” signifies the third lineage-specific whole genome duplication that occurred in teleosts. Amphioxus possess four copies of a gene that is closely related to the GnRH receptors, as illustrated in Figure 5.

Figure 10

Complete open reading frame sequences of representative GnRH receptors, aligned using Clustal X [ 58 ]. Arrowheads indicate intron-exon boundaries. Locations of transmembrane domains (TM) are estimated based on those illustrated in [25] and in [10]. a = GnRH binding site for Type I receptors [59]; b = G_s coupling site in Type I receptors [24]; c = GnRH2 binds receptors containing D at this position, and does not bind receptors containing an N at this position [56]; d = GnRH binding site in both Type I and II receptors [24,55]; e = G_q/11 coupling site in Type I receptors [24]; f = GnRH binding site in Type II receptors [55]; g = sites contributing to differential sensitivity to GnRH1 and GnRH2 [57]; h = site involved in activation of the adenylyl cyclase/protein kinase A signaling pathway [60]; i = site involved in rapid internalization of Type II receptors [61].