Distinct expression patterns of glycoprotein hormone subunits in the lophotrochozoan Aplysia: implications for the evolution of neuroendocrine systems in animals

Abstract

Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

Fig. 1.

A, Amino acid sequence of the newly identified A. californica GPHβ (AcaGPHβ) subunit and B) AcaGPHα subunit with other GPH subunits. For AcaGPHβ (A) only four of the six disulfide bonds found in human hCG (HomohCG) β-subunit are conserved in Aplysia, Drosophila, and human GPHβ5. Numbers in the header row indicate cysteines involved in disulfide bonds for human hCG. Stars indicate conservation of these cysteines across all species aligned. Note that there are six predicted disulfide bridges in human GPHβ subunits (here represented by hCG) and five in human GPHα1 subunit. Dashed lines in the footer row indicate hairpin loops (HP1 and HP2) for GPHβ5. Solid line with arrows marks the position of the long loop (LL) for GPHβ5. Shading marks degree of conservation of residues. Note that conservation between GPHα2 and GPHα1 was too low to predict specific structural features from the alignment (B). HomohCG, Homo sapiens hCG: Uniprot, P01233; HomoGPHβ5, H. sapiens glycoprotein β 5 subunit: Uniprot, Q86YW7; Drosophila GPHβ5, D. melanogaster GPH β 5 subunit: Uniprot, Q8MLY9; HomoGPHα1, H. sapiens GPH α subunit: Uniprot, P01215; DrosophilaGPHα2, D. melanogaster glycoprotein α 2 subunit: Uniprot, Q58L88; HomoGPHα2, H. sapiens GPH α 2 subunit: Uniprot, Q96T91; AcaGPHβ, A. californica GPH β subunit; AcaGPHα, A. californica GPH α subunit.

Fig. 2.

The 3D reconstruction of AcaGPHβ subunit confirms conserved GPH structure and reveals putative new structural elements. A, We used the crystal structure of hCG β subunit (A: hCG: Uniprot P01233; PDB 1HCN) and aligned the AcaGPHβ (B) sequence manually to it in PYMOLTM v. 0.99rc6. The same method was used to model human Thyrostimulin β subunit (C) (HsGPHβ5: Uniprot Q86YW7), an ortholog of the AcaGPHβ subunit. Both hairpin loops (HP1 and HP2) as well as the long loop (LL) appear to be structurally largely conserved in Aplysia with the exception that the model used predicts an α-helix in hairpin loop 1 potentially involved in receptor and/or α-subunit interaction. Arrows point to disulfide bonds (indicated by yellow cylinders) that form the cysteine knot. Both proteins are predicted to form four disulfide bonds. In hCG (A) the heterodimer between α- and β-subunit is stabilized by a seatbelt sequence. This structure is buckled by a disulfide bridge formed between cysteine residues 3 (positions 26 and 110) marked in Fig. 1 and shown in panel A. Note that these cysteine residues are missing in AcaGPHβ subunits (B) and human GPHβ5 (C), and it is therefore assumed that a seatbelt and buckle structure is not formed.

Fig. 3.

ML and BMCMC Bayesian phylogenetic analysis of metazoan GPHα and GPHβ subunits. ML topology shown here. Nodal support metrics are given by bootstrap proportions of likelihood (above branch) and Bayesian posterior probabilities (below branch) and are depicted as circles. Yellow more than 50; green more than 60; blue more than 80; red more than 90; White = 100. See Supplemental Figs, 2 and 3 for original phylogenies. Both isoforms for human GPH α1, α2, and β5, and Strogylocentrotus β5 are included.

Fig. 4.

Phylogenetic relationships of the taxa used for comparative genomic analyses of GPH phylogeny (see Supplemental Table 1). Our phylogenetic results allow the inference of eight well-supported gene duplication events. These include gene duplications that have lead to: 1) the α- and β- subunit paralogy groups in the lineage leading to Bilateria; 2) the α1- and α2-subunits in the lineage leading to vertebrates; 3) the β5 and GC, LH, FSH, and TSH-clade ancestor in the lineage leading to vertebrates; 4) the TSH and FSH paralogs in the lineage leading to amniotes; 5) GC and LH genes in the lineage leading to Homo. Additional duplications and several gene losses must have also occurred in the evolutionary history of the GPH gene family, but these cannot be placed with accuracy given lack of support for certain internal nodes in our phylogeny.

Fig. 5.

Temporal expression patterns of AcaGPHβ and AcaGPHα mRNA during development (A) show increased abundance of mRNA levels in larval stages during larval stages and a strong correlation between α- and β-subunits. All values were calculated as relative expression levels to the gastrula stage using ribosomal protein S5 from Aplysia as a control gene. Therefore, the expression levels for the relative expression levels for the gastrula stage are set at 0 in this diagram and not shown. Schematic representation of juvenile nervous system shows maturation of the ganglionic CNS with respect to developmental stages. B, representative developmental stages of Aplysia show stages tested for qRT-PCR in panel A. Cl, Cleavage stages; Tr, Trochophore stage; Tr-Vel, Trochophore-veliger transition; PreH, prehatching; preM, pre-metamorphic/metamorphically competent; PostM: post-metamorphosis.

Fig. 6.

Spatial expression patterns of AcaGPHα and AcaGPHβ mRNA in ganglia of the CNS of A. californica detected with antisense probe using ISHs. For detailed quantitative expression data see Table 1. A, Schematic representation of ganglionic nervous system of Aplysia CNS indicating major ganglia: BG, buccal ganglia; CG,cerebral ganglia; PG, pedal ganglia; PlG, pleural ganglia; AG, abdominal ganglia. B, Rostral surface of buccal ganglion (BGr) showing AcaGPHβ localization in cells in proximity of buccal sensory cells (circle). B′, Caudal surface of buccal ganglion (BGc) showing highly specific localization of AcaGPHβ staining in three cells that have not yet been described. B″, Rostral surface of buccal ganglion showing abundant localization of AcaGPHα. C, Dorsal surface of cerebral ganglion (CGd) showing AcaGPHβ staining in various yet unidentified cells. MCC and CPC (cerebropedal connectivity) are given as landmarks. Circles point out both F and A/B cluster, which are both referred to in the text. Note that no staining was localized in any of these cell clusters. C′, Ventral surface of cerebral ganglion (CGv) showing AcaGPHβ staining. MCC cells are indicated as landmarks. Very few cells were identified on the ventral side of the cerebral ganglion (arrows). C″, Partial view of dorsal side of cerebral ganglion (CGd) showing GPHα2 staining. None of these cells could directly be associated with any known cell type in the cerebral ganglia. Position of MCC cells is given as a landmark. D, Dorsal view of left pleural ganglion (PlGd) stained for AcaGPHβ subunit mRNA. In addition to a few unknown cells in the central part of the ganglia, the main staining is localized in the pleural sensory cell cluster (circle). Note that this cluster is also staining for AcaGPHα probe (E′ and E″). D′, Ventral view of left pleural-pedal ganglion (PlGv) stained with AcaGPHα antisense probe. Staining occurs in pleural sensory cluster (circle) and some associated cells that have not been previously identified. Note that this staining is highly similar to the staining of the GPHβ5 subunit seen in panel E. D″, Dorsal view of pedal-plural ganglion (PG+PlGd) stained with AcaGPHα antisense probe shows positive staining in the pleural sensory cluster. Note that this staining is highly similar to the staining of the AcaGPHβ subunit seen in panel E. PlPC (pedalpleural connectivity) and nerves P5 and P9 are given as landmarks. E, Ventral view of abdominal ganglia (AGv) showing expression of AcaGPHβ in unknown cells (arrows). Note that these cells also express AcaGPHα as seen in E′. Nerve A2 is given as a landmark. E′, Ventral view of abdominal ganglion showing expression of AcaGPHα (arrows). Note that these cells also express AcaGPHβ as seen in E. Nerve A2 is given as a landmark. E″, Dorsal view of abdominal ganglion shows asymmetric expression of AcaGPHα (arrows).