Glycoprotein hormones and their receptors emerged at the origin of metazoans

Abstract

The cystine knot growth factor (CKGF) superfamily includes important secreted developmental regulators, including the families of transforming growth factor beta, nerve growth factor, platelet-derived growth factor, and the glycoprotein hormones (GPHs). The evolutionary origin of the GPHs and the related invertebrate bursicon hormone, and their characteristic receptors, contributes to an understanding of the endocrine system in metazoans. Using a sensitive search method with hidden Markov models, we identified homologs of the hormones and receptors, along with the closely related bone morphogenetic protein (BMP) antagonists in basal metazoans. In sponges and a comb jelly, cystine knot hormones (CKHs) with mixed features of GPHs, bursicon, and BMP antagonists were identified using primary sequence and phylogenetic analysis. Also, we identified potential receptors for these CKHs, leucine-rich repeat-containing G protein-coupled receptors (LGRs), in the same species. Cnidarians, such as the sea anemone, coral, and hydra, diverged later in metazoan evolution and appear to have duplicated and differentiated CKH-like peptides resulting in bursicon/GPH-like peptides and several BMP antagonists: Gremlin (Grem), sclerostin domain containing (SOSD), neuroblastoma suppressor of tumorigenicity 1 (NBL1), and Norrie disease protein. An expanded cnidarian LGR group also evolved, including receptors for GPH and bursicon. With the appearance of bilaterians, a separate GPH (thyrostimulin) along with bursicon and BMP antagonists were present. Synteny indicates that the GPHs, Grem, and SOSD have been maintained in a common gene neighborhood throughout much of metazoan evolution. The stable and highly conserved CKGFs are not identified in nonmetazoan organisms but are established with their receptors in the basal metazoans, becoming critical to growth, development, and regulation in all animals.

Keywords: BMP antagonist; GPH evolution; LGR evolution; bursicon; cystine knot growth factor; thyrostimulin.

Fig. 1.—

Alignment of superfamily cystine knot growth factor (CKGF) groups from metazoa. (A) An overview of superfamily members showing the core cysteines as a black box with a white “C.” The conserved glycine is in a gray box marked “G.” The six cysteines that form the knot are numbered below the diagram with the three conserved cystine knot disulfide bridges shown by connecting arrows. (B) Amino acid alignment of the cystine knot domain from selected families in (A), followed by partial C-terminal tail. (C) The N-terminal region of the proteins shown in (B). The predicted signal peptides (SP) are highlighted in gray and putative N-linked glycosylation sites are highlighted in dark blue. The individual families are shown by light blue bars for glycoprotein hormones (GPH): α1 (common subunit for LH, FSH, TSH, and hCG), β1-4 (subunits for LH, FSH, TSH, and hCG), α2, and β5 (subunits for thyrostimulin). Medium blue bars represent bursicon (Burs) hormones with α subunits and β subunits. The orange bar shows the cnidarian (cnid.) hormone that has features of both bursicon and GPHs (Burs/GPH). Placozoans also have putatie peptides with the Burs/GPH primary structure. The yellow bars show the comb jelly cystine knot hormone (CKH) and the sponge (three species) CKHs. The green bars represent different families known as BMP antagonists: Norrie disease protein (NDP), gremlin (Grem), neuroblastoma suppressor of tumorigenicity 1, and sclerostin domain containing (SOSD). The white bar shows the transforming growth factor β (TGFβ); the light gray bar is platelet derived growth factor (PDGF); and the dark gray bar is nerve growth factor (NGF).

Fig. 1.—

Alignment of superfamily cystine knot growth factor (CKGF) groups from metazoa. (A) An overview of superfamily members showing the core cysteines as a black box with a white “C.” The conserved glycine is in a gray box marked “G.” The six cysteines that form the knot are numbered below the diagram with the three conserved cystine knot disulfide bridges shown by connecting arrows. (B) Amino acid alignment of the cystine knot domain from selected families in (A), followed by partial C-terminal tail. (C) The N-terminal region of the proteins shown in (B). The predicted signal peptides (SP) are highlighted in gray and putative N-linked glycosylation sites are highlighted in dark blue. The individual families are shown by light blue bars for glycoprotein hormones (GPH): α1 (common subunit for LH, FSH, TSH, and hCG), β1-4 (subunits for LH, FSH, TSH, and hCG), α2, and β5 (subunits for thyrostimulin). Medium blue bars represent bursicon (Burs) hormones with α subunits and β subunits. The orange bar shows the cnidarian (cnid.) hormone that has features of both bursicon and GPHs (Burs/GPH). Placozoans also have putatie peptides with the Burs/GPH primary structure. The yellow bars show the comb jelly cystine knot hormone (CKH) and the sponge (three species) CKHs. The green bars represent different families known as BMP antagonists: Norrie disease protein (NDP), gremlin (Grem), neuroblastoma suppressor of tumorigenicity 1, and sclerostin domain containing (SOSD). The white bar shows the transforming growth factor β (TGFβ); the light gray bar is platelet derived growth factor (PDGF); and the dark gray bar is nerve growth factor (NGF).

Fig. 2.—

Phylogenetic tree of cystine knot hormones (CKHs) and homologs in basal metazoans. Members of the cystine knot growth factor (CKGF) superfamily were aligned, degapped, and trimmed to include only the cystine knot domain; a maximum-likelihood phylogeny is shown with SH-like supports and Bayesian posterior probabilities, respectively. All cnidarian sequences (sea anemone and coral) are identified with an adjacent arrow. The individual families are shown by light blue bars for glycoprotein hormone (GPH): α2 and β5 (subunits for thyrostimulin). Medium blue bars represent bursicon (Burs) hormones with α subunits and β subunits. The orange bar shows the cnidarian hormone that has features of both bursicon and GPHs (Burs/GPH). The yellow bars show the comb jelly CKH and the sponge (three species) CKHs. The green bars represent different families known as bone morphogenetic protein (BMP) antagonists: Norrie disease protein (NDP), gremlin (Grem), neuroblastoma suppressor of tumorigenicity 1 (NBL1), and sclerostin domain containing (SOSD). The scale bar denotes amino acid substitutions per site and the arrows indicate cnidarian sequences. The tree was rooted to a group of bilaterian, comb jelly, and sponge transforming growth factor β (TGFβ) family sequences. Maximum-likelihood trees were constructed with RAxML 7.7.9 under the PROTGAMMAIWAG model, and the best tree topology was selected with CONSEL 0.20 using the AU statistic. This tree is presented, with SH-like support values first and posterior probability support values second, from a separate Bayesian inference tree constructed with PhyloBayes MPI 1.4f under similar conditions (estimated gamma, WAG substitution model). Supplementary trees, the alignment, and the sources of sequences used are found in supplementary figure S2, Supplementary Material online.

Fig. 3.—

Phylogenetic tree of leucine-rich repeat-containing G protein-coupled receptors (LGRs) in basal metazoans. LGRs, including those of basal metazoans (cnidarians such as anemone and corals, comb jelly, and sponges) and homologs from several bilaterian species, were aligned, degapped, and trimmed to include only the seven transmembrane domain. A maximum-likelihood tree was constructed with RAxML 7.7.9 under the PROTGAMMALGF model. This tree is presented in compressed format, with bootstrap support values first and posterior probability support values second, from a separate Bayesian inference tree constructed with PhyloBayes MPI 1.4f under similar conditions (estimated gamma, LG substitution model). The scale bar denotes amino acid substitutions per site. The tree was rooted to a group of bilaterian opsins and neuropeptide G protein coupled receptors. Supplementary trees, the alignment, and the sources of sequences used are found in supplementary figure S3, Supplementary Material online. Annel., annelid; CKHR, cystine knot hormone receptor; FSHR, follicle-stimulating hormone receptor; GPHR, glycoprotein hormone receptor; GRL101, LGR with multiple LDLa domains; LHR, luteinizing hormone receptor; Mol., mollusc; Thyrostim. R, thyrostimulin receptor; TSHR, thyroid-stimulating hormone receptor.

Fig. 4.—

Primary sequence domains found in basal metazoan leucine-rich repeat-containing G protein-coupled receptors (LGRs). Domains common to the fruit fly glycoprotein hormone receptor (GPHR, also knows as thyrostimulin receptor) and fly bursicon receptor (BursR) are compared with receptors predicted from the sea anemone (Nematostella vectensis), comb jelly (Mnemiopsis leidyi), and a sponge (Ephydatia muelleri). The signal peptide (SP, red), leucine-rich repeats (LRRs, yellow), “hinge” (green) region, and seven transmembrane (7TM) domain (gray) are displayed, along with the C-terminal tail, approximately to scale. The sequences used, with these features highlighted, can be found in supplementary figure S4, Supplementary Material online.

Fig. 5.—

Synteny of genes encoding GPHα2/GPHβ5, gremlin (Grem), and sclerostin domain containing (SOSD) in human, amphioxus, limpet, and sea anemone. The diagram shows a representation of the common ortholog neighborhood shared by the genes encoding the thyrostimulin subunits (GPHα2/GPHβ5), shown in blue, and two bone morphogenetic protein (BMP) antagonists, Grem and SOSD, shown in green. Orthologs common to humans, amphioxus (Branchiostoma floridae, JGI assembly v2), the limpet (Lottia gigantea, JGI assembly v1), and the sea anemone (Nematostella vectensis, JI assembly v1) are shown in gray if they belong to the ancestral chordate linkage group 11, or pink if not. Genes are ordered according to how they are found on the limpet scaffolds, with their species-specific location in megabases (Mb) listed under the corresponding gene box. The orthologs of the genes encoding GPHα2/GPHβ5 found in the sea anemone are labeled B/GPH for bursicon/glycoprotein hormone. All gene boxes are labeled with their uppercase protein designations for legibility.

Fig. 6.—

Detection of cystine knot growth factor (CKGF) and leucine-rich repeat-containing G protein-coupled receptor (LGR) homologs in eukaryotic groups. Members of the CKGF superfamily are listed across the top of the diagram, with dots indicating the presence (colored) or absence (white) of orthologs in each metazoan or nonmetazoan group. A representative phylogeny of the various metazoan and nonmetazoan groups is shown on the left, with branches color-coded according to the presence of cystine knot hormone (CKH, yellow), bursicon/glycoprotein hormone (B/GPH, orange) glycoprotein hormones (GPHs, blue), and bursicon (Burs, blue). Arrows indicate the ancestral node where the indicated peptides presumably originated. LGRs are listed in red at the top right of the diagram; they are the presumptive Type A, B, or A/B receptors for the aforementioned peptides. Metazoan and nonmetazoan databases were searched for the presence of homologs, and all major sequenced groups of amorpheans are displayed. Specific species databases that were searched are listed in supplementary table S1, Supplementary Material online. BMP ant., bone morphogenetic protein antagonist; NGF, nerve growth factor family; PDGF, platelet-derived growth factor family; TGFβ, transforming growth factor beta family.