Widespread divergence of the CEACAM/PSG genes in vertebrates and humans suggests sensitivity to selection.

In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs) and pregnancy-specific glycoproteins (PSGs) play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal-maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture-including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif-are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs) at the CEACAM/PSG locus. These copy number polymorphisms have 3-80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP) compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal-fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.

Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs.

Relaxin peptides are important hormones for the regulation of reproductive tissue remodeling and the renal cardiovascular system during pregnancy. Recent studies demonstrated that two of the seven human relaxin family peptides, relaxin H2 (RLN2) and INSL3, signal exclusively through leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8. Although it was well characterized that an RXXXRXXI motif at the RLN2 B chain confers receptor activation activity, it is not clear what roles RLN2 A chain plays in receptor interaction. Analyses of relaxin family genes on syntenic regions of model tetrapods showed that the A chain of RLN2 orthologs exhibited a greater sequence divergence as compared with the receptor-binding domain-containing B chain, foreshadowing a potential role in receptor interactions; hence, defining receptor selectivity in this fast evolving peptide hormone. To test our hypothesis that select residues in the human RLN2 A chain play key roles in receptor interaction, we studied mutant peptides with residue substitution(s) in the A chain. Here, we showed that alanine substitution at the A16 and A17 positions enhances LGR8-activation activity of RLN2, whereas mutation at the A22-23 region (RLN2A22-23) ablates LGR8, but not LGR7, activation activity. In addition, we demonstrated that the functional characteristics of the RLN2A22-23 mutant are mainly attributed to modifications at the PheA23 position. Taken together, our studies indicated that ThrA16, LysA17, and PheA23 constitute part of the receptor-binding interface of human RLN2, and that modification of these residues has led to the generation of novel human RLN2 analogs that would allow selective activation of human LGR7, but not LGR8, in vivo.

Origin of INSL3-mediated testicular descent in therian mammals.

Testicular descent is a unique physiological adaptation found in therian mammals allowing optimal spermatogenesis below core body temperature. Recent studies show that INSL3, produced by Leydig cells, and its receptor LGR8 (RXFP2) are essential for mediating the transabdominal phase of testicular descent during early development. However, the origin and genetic basis for this physiological adaptation is not clear. Using syntenic mapping and the functional characterization of contemporary and resurrected relaxin family hormones, we show that derivation of INSL3-mediated testicular descent involved the duplication of an ancestral RLN3-like gene that encodes an indiscriminate ligand for LGR7 (RXFP1) and LGR8. This event was followed by acquisition of the LGR7-selective characteristics by a daughter gene (RLN3) prior to the evolution of the common ancestor of monotremes, marsupials, and placentals. A subsequent mutation of the other daughter gene (INSL3) occurred before the emergence of therian mammals, which then led to the derivation of the reciprocal LGR8-specific characteristics of INSL3. The stepwise evolution of these independent signaling pathways through gene duplication and subsequent divergence is consistent with Darwinian theory of selection and adaptation, and the temporal proximity suggests an association between these genetic events and the concurrent evolution of testicular descent in ancestral therian mammals.

GPCR genes are preferentially retained after whole genome duplication.

One of the most interesting questions in biology is whether certain pathways have been favored during evolution, and if so, what properties could cause such a preference. Due to the lack of experimental evidence, whether select gene families have been preferentially retained over time after duplication in metazoan organisms remains unclear. Here, by syntenic mapping of nonchemosensory G protein-coupled receptor genes (nGPCRs which represent half the receptome for transmembrane signaling) in the vertebrate genomes, we found that, as opposed to the 8-15% retention rate for whole genome duplication (WGD)-derived gene duplicates in the entire genome of pufferfish, greater than 27.8% of WGD-derived nGPCRs which interact with a nonpeptide ligand were retained after WGD in pufferfish Tetraodon nigroviridis. In addition, we show that concurrent duplication of cognate ligand genes by WGD could impose selection of nGPCRs that interact with a polypeptide ligand. Against less than 2.25% probability for parallel retention of a pair of WGD-derived ligands and a pair of cognate receptor duplicates, we found a more than 8.9% retention of WGD-derived ligand-nGPCR pairs--threefold greater than one would surmise. These results demonstrate that gene retention is not uniform after WGD in vertebrates, and suggest a Darwinian selection of GPCR-mediated intercellular communication in metazoan organisms.

Conservation of the heterodimeric glycoprotein hormone subunit family proteins and the LGR signaling system from nematodes to humans.

Glycoprotein hormones, follicle-stimulating hormones (FSHs), luteinizing hormones (LHs), thyroid-stimulating hormones (TSHs), and chorionic gonadotropin (CG) are key endocrine hormones secreted from the pituitary gonadotrophs and thyrotrophs and the placenta in primates. These hormones, consisting of a common alpha subunit and a specific beta subunit, act through the FSH receptor (FSHR), the LH receptor (LHR), and the TSH receptor (TSHR) that are highly specific for their cognate hormones. These glycoprotein hormones are structurally and functionally conserved in various vertebrates and have been identified in most lineages of actinopterygians (bony fish) and sarcopterygians (tetra-pods). Of interest, recent genomic studies showed that vertebrate glycoprotein hormone receptors belong to an ancient subfamily of G protein-coupled receptors (GPCRs) named as leucine-rich repeat-containing GPCRs (LGRs). These findings have prompted the hypothesis that there could be additional glycoprotein hormones in vertebrate genomes. Indeed, searches of vertebrate genomes have led to the identification of two novel glycoprotein hormone subunits, glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5), as well as their homologs in invertebrates. Subsequently, it was demonstrated that GPA2 and GPB5 form a heterodimeric hormone, thyrostimulin/OGH, capable of activating TSHR in vivoand the thyroid axis in transgenic mice. However, the exact role of this novel glycoprotein hormone and its homolog in invertebrates is not clear. To gain a better understanding of the physiological role of the novel glycoprotein hormone subunits and their evolution, it is imperative to carry out systematic studies of these genes in representative model species. In the present report, we summarize our findings based on studies of genomes of model organisms from sea anemones to humans. We found that GPA2 and GPB5 represent the ancient forms of glycoprotein hormone alpha and beta subunits, respectively, and that vertebrate and invertebrate glycoprotein hormone subunit proteins shared common ancestors that evolved during early metazoan evolution. It is important to note that glycoprotein hormone alpha and beta subunit proteins from invertebrates formed a heterodimer with structural functional characteristics similar to that of vertebrate glycoprotein hormones. Taken together, both glycoprotein hormone alpha and beta subunits evolved before the evolution of nematodes, arthropods, and vertebrates.

Evolution of the signaling system in relaxin-family peptides.

Recent studies have characterized two G-protein-coupled receptors (GPCRs), LGR7 and LGR8, as relaxin receptors. Later studies have shown that LGR7 and LGR8 also are cognate receptors for the relaxin-family peptides, INSL7/relaxin3 and INSL3, respectively. In addition, INSL7/relaxin3 signals through two orphan GPCRs, GPCR135 and GPCR142, whereas INSL5 is a select ligand for GPCR142. These findings have greatly enhanced our understanding of the physiology and signaling of this unique group of peptide hormones. Phylogenetic analysis of relaxin-family peptides and their co-evolved receptors suggests that the ancestor relaxin gene duplicated multiple times in a vertebrate branch-specific manner. Among the seven human relaxin-family peptides (relaxin1, relaxin2, INSL3/RLF, INSL4/EPIL, INSL5/RIF2, INSL6/RIF1, and INSL7/relaxin3), INSL7 and INSL5 could represent the most ancient form. By contrast, the most widely studied family peptides, human relaxins H1 and H2, appear to be derived from recent gene duplication in mammals. Therefore, relaxin-family peptides could be important for the evolution and adaptation to lineage-specific physiologic processes during evolution. Duplicated relaxin-family genes assumed regulatory roles in newly evolved reproductive processes, and relaxin/LGR signaling was harnessed for signaling in the uterus and mammary gland in addition to other tissues. Although the precise evolutionary history of relaxin ligand/receptor pairs remains to be elucidated, these findings indicate that the expansion of relaxin-family genes and their specific regulatory functions have evolved during vertebrate evolution to allow the development of a tissue-specific regulatory mechanism in a lineage-specific manner and provide a revealing portrait of molecular evolution in action.